Cancer stem cells and uses thereof

ABSTRACT

Disclosed are enriched preparations of neuroblastoma tumor initiating cells (NB TICs). The NB TICs are capable of self-renewal, initiating neuroblastoma tumor growth in vivo and are capable of being passaged in high frequency. These NB TICs have chromosomal abnormalities and are capable of giving rise to secondary tumor spheres. Methods are also disclosed for preparing the enriched preparations of NB TICs, such as from neuroblastoma tumor tissue and metastasized bone marrow. Also disclosed are methods of screening candidate substances to identify therapeutic agents for the treatment of neuroblastoma. Methods are also provided for screening a sample for neuroblastoma, as well as for screening a sample to identify the stage of neuroblastoma present. Kits are also provided for selecting appropriate anti-neuroblastoma compounds for a patient, and utilize isolated compositions of the patients&#39; neuroblastoma tumor initiating cells. In this manner, a customized medicinal profile for the patient may be devised.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/562,798, filed Nov. 22, 2009, which makes reference to thefollowing provisional U.S. Patent Application No. 60/739,337 entitled“Cancer Stem Cells and Uses Thereof”, filed Nov. 23, 2005. The entiredisclosure and contents of the above application is hereby incorporatedby reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to preparations of stem cells,particularly cancer stem cells derived from neural crest tissue. Theinvention also relates to methods of isolating cancer stem cells, andvarious methods for using cancer stem cells in diagnostic, therapeuticand other clinical and non-clinical applications.

2. Related Art

Neuroblastoma (NB) is the most common extracranial solid tumors inchildren, with poor survival rates in children with metastatic disease.NB is estimated to be responsible for about 15% of cancer-related deathsin children (1, 2). The survival rate for metastatic NB is estimated tobe less than 30%. In the majority of these cases, conventional cancertherapies have been ineffective.

Little is reported concerning the precise molecular alterations thatgive rise to NB, its cell of origin, or why NB cells metastasize andbecome resistant to chemotherapeutic agents. Unfortunately, geneticmutations that contribute to the origin and progression of 98% of NBcases have not been identified.

One identifiable hallmark of NB is the appearance of proliferating cellswith characteristics of neural crest-derived sympathetic neuronalprecursors (neuroblasts). NB tumors also frequently contain other neuralcrest cell types, including neuroendocrine and Schwann cells. Moreover,NB appears in tissues that developmentally derive from the neural crestincluding sympathoadrenal precursors which differentiate into bothsympathetic neurons and adrenal chromaffin cells, the paravertebral andpreaortic sympathetic ganglia, and the adrenal gland.

The clinical behavior of NB is unique. Tumors that arise in childrenunder one year of age may spontaneously regress by differentiation orapoptosis, even after arising in or metastasizing to liver and skin. Incontrast, NB tumors in children over one-year-old often growaggressively, disseminate to the bone and bone marrow, and are fatal inthe vast majority of cases.

Mass screening of infants showed that NB is much more frequent thanpreviously thought. Many of these tumors regress without clinicaldiagnosis. Regressing or favorable-prognosis tumors have been reportedto express high levels of the TrkA/NGF receptor and display phenotypesof differentiated peripheral neural cells, while malignant orunfavorable-prognosis tumors resemble proliferating sympathoadrenalprecursors, often expressing TrkB, amplified MYCN, and many genesinvolved in neural crest development.

The only reported germline NB predisposition gene is Phox2b, which ismutated in many familial cases of NB, and is required for properdifferentiation of sympathetic neurons from neural crest precursors(NCPs) (3,4). In the regressive form of the disease, the precursor cellsultimately differentiate or die, while in older children, thesemolecular transformations instead result in a population of persistentlyproliferating and highly migratory transformed neuroblasts.

The concept of tumor-initiating cells (TIC) (also called tumor or cancerstem cells) postulates that only rare cells in tumors are endowed withtumorigenic potential, and was initially developed to explain why (i)most tumors are comprised of both undifferentiated proliferatingprogenitors and post mitotic differentiated cells, (ii) only a verysmall fraction of tumor cells form colonies after plating in vitro, and(iii) large numbers of tumor cells are required to seed the growth of anew tumor in mice (4-10).

Dick et al. and others reported that clonally-derived tumor cells ofacute myelogenous leukemia (AML) patients could be physically separatedinto tumorigenic and non-tumorigenic fractions (11, 12). Brain andbreast tumors have also been reported to contain a subpopulation of TICs(13, 14). Thus, in solid tumors, a rare tumor cell population may fueltumor growth and seed metastasis. This hypothesis has major implicationsfor treating cancer patients. For example, many current therapies killthe bulk of proliferating tumor cells, but these cells may not beintrinsically tumorigenic, and in many cases the TICs may escape theeffects of the therapeutic agents, leading to tumor relapse. Thus, it isessential to identify and characterize TICs from various tumors in orderto develop and target therapies against this critical cell type.

TICs have also been shown to share phenotypic characteristics with stemcells derived from their tissue of origin. For example, for a giventissue, the tissue stem cells and TICs both (i) self-renew, (ii) expresscommon phenotypic markers, (iii) grow in a similar fashion in responseto mitogens, and (iv) yield tissue-appropriate progeny (13, 14).However, whereas tissue stem cells generate mature differentiated celltypes, differentiation of TICs is generally arrested at the level of oneor more tissue progenitor cells resulting in tumors comprising ahierarchy of progenitors and some differentiated progeny (4).

Many pediatric and adult tissues contain resident stem cells (4). It iscurrently unknown if TICs originate by transformation of tissue stemcells. Observations have been made that oncogenic mutations commonlyaffect genes required for normal stem cell renewal and differentiation(4). This may be particularly relevant for children's tumors, sincedeveloping tissues contain a higher proportion of tissue stem cells thando adult tissues.

Tumor initiating cells from some solid tissue tumors, such as breast andbrain tumors, have been described. However, a TIC population from tumortissue in a patient with NB has not been isolated. One reportedobservation in some infantile forms of NB (called stage 4S) is thatlarge tumors are frequently found in skin (15). It was previouslyassumed that skin was a preferred metastatic target for NB. However, apopulation of TICs from such solid tumor tissue has not yet beenreported.

The above and other observations in the field reveal a continuingmedical need continues to exist in the art to determine why and in whichcell type NB arises, and why some NB tumors spontaneously regress andothers are fatal. In addition, new effective drug targets andtherapeutics tailored to identifying and treating specific forms andstages of NB are needed.

SUMMARY

The above and other long felt needs in the art are met in the presentinvention.

The present invention demonstrates the isolation and preparation, aswell as uses of, a unique population of tumor initiating cells (TICs)that are characteristic of a condition known as neuroblastoma (NB). Itis from these preparations that the various compositions, methods ofuse, screening methods and therapeutic treatments described herein areprimarily provided.

Tumor Initiating Cells/Neuroblastoma Tumor Initiating Cells

In one aspect, the invention provides a composition comprising tumorinitiating-cells, and in particular, neuroblastoma tumor initiatingcells (NB TICs). Therefore, and in accordance with the first broadaspect of the present invention, there is provided a compositioncomprising an enriched population of cancer stem cells comprising NBTICs.

An enriched population of NB TICs may be defined in some aspects of theinvention as comprising a population of NB TICs that is greater than theconcentration of tumor initiating NB cells in a non-concentrated NBtissue preparation, such as in a NB tumor tissue or bone marrow tissuepreparation prepared from a tissue obtained from an animal having NBwithout tissue and/or culture processing.

The enriched preparations of the invention may further be described as afunction of the percentage of NB TICs capable of giving rise to asecondary NB sphere in culture contained in the preparation. By way ofexample, the percentage of a starting NB tumor cell population that iscapable of giving rise to secondary NB tumor spheres in a non-enrichedpreparation of NB tumor tissue is about 0.2% to 2.0%. In an enrichedpreparation or composition, the percentage of NB tumor cells present inthe composition that are capable of giving rise to secondary NB tumorspheres is greater than 2%, and in some embodiments, from 3% to about18%, of the total cell population contained in the preparation.

The enriched preparations of the invention may further be described as afunction of the percentage of NB TICs capable of giving rise to a tumoras compared to the non-enriched population. In an enriched preparationor composition, the percentage of NB tumor cells present in thecomposition that are capable of giving rise to an NB tumor is 1 in 100,or 1% of the total cell population contained in the preparation.

The NB TIC preparations are further described as capable of initiatingthe growth of a NB tumor in vivo. The enriched preparations of theinvention may further be described as a function of the percentage of NBTICs capable of giving rise to a tumor as compared to the non-enrichedpopulation. By way of example, the percentage of a non-enrichedpopulation or of an NB established cell line that is capable of givingrise to a tumor is 1 in 2×10⁶ cells in a commonly used NB mousexenograph model (28) In an enriched preparation or composition, thepercentage of NB tumor cells present in the composition that are capableof giving rise to an NB tumor is 1 in 100, or 1% of the total cellpopulation contained in the preparation.

The NB TIC preparations are further described as capable of initiatingthe growth of a NB tumor in vivo when enriched for the cell-surfaceproteins CD24 and CD34. The further enriched preparations of theinvention will, when injected into a commonly used neuroblastoma mousexenograph model (28), cause morbidity at twice the rate of populationsof NB TICs not expressing both CD24 and CD34.

The NB TIC preparations may also be further described as comprisingpostnatal neural crest precursor (NCP) cells. In some embodiments, theseNCP cells comprise Skin-derived Precursor (SKP) cells.

In some embodiments, the composition comprises NB TICs derived fromhuman tissue, such as tissue obtained from a tumor or needle biopsy. Byway of example, the human tissue may comprise bone marrow tissue ordermis tissue. In some embodiments, the bone marrow tissue or dermistissue is further described as tissue derived from an infant or child.

The NB TICs of the present invention and compositions and/orpreparations that include them are further described as having thefollowing characteristics: isolated directly from patients withneuroblastoma, express cell surface markers that are characteristic ofdeveloping neural crest stem cells, are capable of forming tumors,particularly NB tumors, in vivo, are self-renewing, are capable of beingpassaged at a high frequency, as having chromosomal abnormalitiescharacteristic of NB, and/or are capable of differentiating into cellswith the properties of sympathetic neurons.

According to yet another broad aspect of the invention, there isprovided a composition comprising an enriched population of cancer stemcells comprising cells from a Stage 1 NB, Stage 2 NB, a Stage 3 NB, aStage 4 NB, or any combination of a Stage 1, Stage 2, Stage 3, and/orStage 4 NB.

Method of Preparing, Isolating and Enriching a Population ofNeuroblastoma Tumor Initiating Cells.

In another broad aspect of the invention, there is provided a method forproviding an enriched population of TICs that more specifically giverise to NB TICs. In some embodiments, the method comprises isolating NBTICs from metastasized bone marrow tissue or from NB tumor tissue, andprocessing said NB TICs so as to obtain a preparation comprising a 2% ormore concentration of NB TICs capable of giving rise to a secondary NBsphere in culture.

By way of example, the percentage of a starting NB tumor cell populationthat is capable of giving rise to secondary NB tumor spheres in anon-enriched preparation of NB tumor tissue is about 0.2% to 2.0%. Inthe present method, the enriched preparation or composition comprises agreater than 2% concentration of NB TICs that are capable of giving riseto secondary NB tumor spheres. In some embodiments, the method providesfor an enriched preparation that comprises from 3% to about 18% NB TICsthat are capable of giving rise to a secondary NB sphere in culture.

In particular embodiments, the invention provides for a method thatcomprises isolating NB TICs from metastasized bone marrow tissue, ratherthan from a NB tumor itself.

The NB TICs may be further characterized as capable of giving rise totumors that are related to the development of the neural crest. By wayof example and not limitation, these types of TICs include those thatgive rise to melanoma, pheochromocytoma, paraganglioma andneurofibromatosis.

In yet another aspect, a screening method is provided for identifyingcandidate anti-cancer therapeutic agents for cancers arising fromdevelopment of the neural crest, including melanoma and other tumorscontaining TICs, such as leukemia, brain, colon and breast. In someembodiments, this screening method employs non-established neural crestcell lines, such as the non-established tumor initiating stem cellsdescribed herein.

Screening Method for Neuroblastoma Tumor Initiating Cell InhibitingCompounds

In another aspect, a screening method is provided comprising a highthroughput screen for identifying candidate anti-NB and/or anti-NB TICtherapeutic agents.

In some embodiments, the method comprises screening a chemical compoundlibrary of interest for activity in a culture comprising an enrichedpreparation of NB TICs. Such a chemical library may include theSPECTRUM™ Collection library, the LOPAC™ Collection library, thePRESTWICK CHEMICAL LIBRARY® and the MAYBRIDGE® Collection library (eachare libraries of compounds for screening).

In some embodiments, the method may be used to screen for a therapeuticagent custom tailored to a specific patient. In some embodiments, themethod in this application would comprise preparing an autogenousculture of NB TICs harvested from an identified patient being screened,and used according to the defined method herein to identify candidatetherapeutic agents anticipated to be effective against the particularpopulation of NB of the identified patient. In this manner, potentialtherapeutic agents may be identified that are without inhibitory orother untoward and/or undesirable effects on normal cell populations ofa patient, such as to a patient's normal stem cell population or immunesystem.

In yet another aspect, a diagnostic screening method is provided usingthe isolated tumor initiating stem cells of the invention. In someembodiments, the method employs the use of NB TIC specific prognosticmarkers to identify the absence or presence of NB, and in some cases thestage of NB, in a biological sample obtained from an animal, such as ahuman. In this manner, the invention also provides for methods ofscreening and identifying particular stages of disease progression in NBstage 1, 2, 3 and 4 diseases, and to identify potential therapeuticagents that may be effective in both identification and treatment.

Nucleic Assay Microarray System

In yet another aspect, a nucleic assay microarray system, such as a cDNAmicroarray method, is provided to identify novel markers and drugtargets for NB stem cells.

Methods of Inhibiting Neuroblastoma Tumor Initiating Cells/Methods ofTreating and/or Inhibiting Neuroblastoma in an Animal

In yet another aspect, the invention provides methods for inhibiting NBTICs. In some embodiments, the method comprises administering aneffective amount of a composition comprising a NB TIC-inhibitingingredient. In some embodiments, the NB TIC inhibiting ingredientcomprises one or more active ingredients comprising:

-   -   2.3-Dimethoxy-1.4-naphthoquinone,    -   Aklavine Hydrochloride,    -   Amodiaquin dihydrochloride dehydrate;    -   Amsacrine Hydrochloride;    -   Azaguanine-8;    -   beta-peltatin;    -   Camptothecine (S.+);    -   CGP-74514A hydrochloride;    -   Chelerythrine chloride;    -   Cholestan-3beta.5alpha.6beta-Triol;    -   Ciclopirox Olamine;    -   Clofazimine;    -   Colchicine;    -   Convallatoxin;    -   Crassin Acetate;    -   Crinamine;    -   Dequalinium analog. C-14 linker;    -   Dequalinium dichloride;    -   Digitoxin;    -   Digoxigenin;    -   Dihydrogambogic acid;    -   Dihydroouabain;    -   Erysolin;    -   Gambogic acid;    -   Mechlorethamine;    -   Meclizine hydrochloride;    -   MG 624;    -   Mitoxanthrone Hydrochloride;    -   Ouabain;    -   Oxybendazole;    -   Oxybendazole;    -   Paclitaxel;    -   Parthenolide;    -   Patulin;    -   Periplocymarin;    -   Peruvoside;    -   Primaquine diphosphate;    -   Quinacrine dihydrochloride;    -   Sanguinarine chloride; or    -   Tomatine.

In some embodiments, the effective amount of the NB TIC-inhibitingingredient is an amount effective to arrest the growth of and/or kill NBTICs, or effective to induce differentiation of said cells to cell typesthat no longer proliferate. In other embodiments, the method may furthercomprise administering a composition further comprising ancitabinehydrochloride, doxorubicin hydrochloride, etoposide, vincristinesulfate, or a combination thereof.

In some embodiments, the compositions may further include apharmaceutically acceptable carrier solution.

In yet other embodiments, the NB TICs are in an animal having NB In someembodiments, the animal is a human. In some embodiments, the human is 12years of age or younger. That is, it is anticipated that the inventionis particularly useful in the treatment of children afflicted with NB,and will have a profound effect on reducing the high rate of mortalityin this population of NB patients.

The method may be further described as administering a composition thathas a reduced non-NB TIC cytotoxicity. It is expected that the methodsand compositions of the present invention will provide fewer and/or lesspronounce undesirable side affect in the treatment of a patient as aresult. In some embodiments, the composition employed in the method isessentially free of non-NB TIC-inhibiting activity.

Kits:

In yet another aspect, the invention provides a kit for the testingand/or screening of a patient of interest's NB TICs. In this manner, asample of biological tissue enriched for a population of NB TICs from apatient of interest may be used to screen and/or identify a specificanti-NB TIC active agent or agents that are the most potent and/oractive against a specific patient's NB TIC population.

In some embodiments, the kit would comprise an assay plate that includesa plurality of wells, each well of said assay plate being suitable forcontaining a pharmacologically active agent of interest, such as apotentially anti-NB TIC pharmacologically active agent. By way ofexample, the assay plate may comprise 40, 50, 60, 70, 80, 90 100 or morewells. In some embodiments, the assay plat will include 96 wells, suchas is customary in assay plates. As part of the kit described herein, 5,10, 20, 25, 30, or 40 of the wells may include a different anti-NB TICcompound, such as a volume of one or more of each of the compoundslisted below:

-   -   2.3-Dimethoxy-1.4-naphthoquinone,    -   Aklavine Hydrochloride,    -   Amodiaquin dihydrochloride dehydrate;    -   Amsacrine Hydrochloride;    -   Azaguanine-8;    -   beta-peltatin;    -   Camptothecine (S.+);    -   CGP-74514A hydrochloride;    -   Chelerythrine chloride;    -   Cholestan-3beta.5alpha.6beta-Triol;    -   Ciclopirox Olamine;    -   Clofazimine;    -   Colchicine;    -   Convallatoxin;    -   Crassin Acetate;    -   Crinamine;    -   Dequalinium analog. C-14 linker;    -   Dequalinium dichloride;    -   Digitoxin;    -   Digoxigenin;    -   Dihydrogambogic acid;    -   Dihydroouabain;    -   Erysolin;    -   Gambogic acid;    -   Mechlorethamine;    -   Meclizine hydrochloride;    -   MG 624;    -   Mitoxanthrone Hydrochloride;    -   Ouabain;    -   Oxybendazole;    -   Oxybendazole;    -   Paclitaxel;    -   Parthenolide;    -   Patulin;    -   Periplocymarin;    -   Peruvoside;    -   Primaquine diphosphate;    -   Quinacrine dihydrochloride;    -   Sanguinarine chloride; or    -   Tomatine.

In addition, and in some embodiments of the kit, at least one or more ofthe assay wells will include a volume of a pharmacologically activeagent that is known and/or is in use as an anti-NB agent, such asancitabine hydrochloride, doxorubicin hydrochloride, etoposide, orvincristine sulfate. In this manner, a positive control is provided inthe assay plate for comparative purposes.

The following abbreviations are used throughout the description of thepresent invention:

-   -   AdGFP—green fluorescent protein expressing adenovirus;    -   AML—acute lymphoblastic leukemia;    -   ARF—ADP-ribosylation factor;    -   BDNF—brain-derived neurotrophic factor;    -   bHLH—basic HLH;    -   BrdU—bromodeoxyuridine;    -   cDNA—copy deoxyribonucleic acid;    -   CNPase—2′3′-cyclic nucleotide 3′-phosphodiesterase;    -   CNS—central nervous system;    -   DRG—dorsal root ganglia;    -   DNA—deoxyribonucleic acid;    -   ΔNp73—N-terminally truncated p73 (lacking the trans-activation        domain)    -   EGF—epidermal growth factor;    -   FACS—fluorescence-activated cell sorting;    -   FGF—basic fibroblast growth factor 2;    -   GalC—galactosyl ceramide;    -   GD2—a disialoganglioside expressed on tumors of neuroectodermal        origin;    -   GFAP—glial fibrillary acidic protein;    -   GFP—green fluorescent protein;    -   GTG-banding—G banding using trypsin and Giemsa;    -   HLH—helix-loop-helix;    -   H&E—hematoxylin and eosin    -   HSC—human stem cell;    -   LOH—loss of heterozygosity;    -   NB—neuroblastoma;    -   NCP—neural crest sympathoadrenal precursors;    -   NFM—neurofilament M;    -   NGF—nerve growth factor;    -   NOD/SKID—nonobese diabetic/severe combined immunodeficient;    -   NT—neural tube;    -   Rb—retinoblastoma;    -   RNA—ribonucleic acid;    -   RT-PCR—reverse transcription-polymerase chain reaction;    -   SCG—sympathetic superior cervical ganglia;    -   shRNA—short hairpin RNA;    -   siRNA—small interfering ribonucleic acid;    -   SKPs—skin-derived precursor cells;    -   SNP—single nucleotide polymorphisms;    -   TH—tyrosine hydroxylase;    -   TIC—tumor initiating cell;    -   TUNEL—terminal deoxynucleotidyltransferase-mediated dUTP nick        end labeling;    -   YFP—yellowish-green fluorescent protein.

In yet another aspect, the invention provides a kit for the testingand/or screening of a patient of interest's tumor-initiating cells(TICs) from tumors such as melanoma, leukemia, brain, breast, and colon.In this manner, a sample of biological tissue enriched for a populationof TICs from a patient of interest may be used to screen and/or identifya specific anti-TIC active agent or agents that are the most potentand/or active against a specific patient's TIC population, using theabove compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in conjunction with the accompanyingdrawings, in which:

FIGS. 1A-1C, according to one embodiment of the invention, relates tothe demonstration of the presence of SKP-like precursors in neonatalmouse adrenal gland and sympathetic ganglia. (1A) Phase illumination ofspheres of proliferating cells obtained from neonatal adrenal gland andsympathetic superior cervical ganglia (SCG). (1B) Double-labelimmunocytochemical analysis of spheres demonstrated that they co expressthe SKP markers, nestin and fibronection. (1C) Double-label analysis ofdifferentiated adrenal gland spheres demonstrated that they gave rise tomorphologically complex cells with the characteristics of sympatheticneurons, such as co expression of neuron-specific βIII-tubulin andneurofilament M (NFM).

FIGS. 2A-2F, according to some aspects of the invention, illustratetumor spheres from various stages of NB. They contain a cell thatproliferates and self-renews under SKP conditions and expresses NB andSKPs markers. (2A) Photomicrographs of human NB tumor spheres generatedfrom bone marrow. (left) and tumor (right) from the same stage 4patient. (2B-2F). Primary spheres from bone marrow aggregates or tumorswere immunostained for the NB markers NB84 (2B, Left panel, lighterregions (red)) and tyrosine hydroxylase (TH) (2B, Right panel, lighterregions (green)) and the SKPs markers nestin (2C, lighter regions(red)), vimentin (2D, lighter regions (red)), fibronectin (2E, lighterregions (green)), and versican (2F, lighter regions (green)).

FIG. 3 A-3C, according to some aspects of the invention, demonstrate(3A) that NB tumor spheres generate sympathetic neuron and Schwann-likecells under neurogenic conditions. Primary spheres were plated onlaminin/lysine coated slides in the absence of growth factors, and after14 days, immunostained for the neuronal markers TH or βIII-tubulin orthe glial markers GFAP or s100β. (3B) Secondary sphere formation fromdissociated NB bone marrow aggregates. The number of spheres formed isproportional to the number of cells plated, and the percent of cellsforming spheres does not vary with the number of plated cells. (3C) NBtumor spheres generate tumors when injected subcutaneously in NOD/SCIDmice. Tumors were excised, sectioned, and immunostained for the NBmarker NB84 (arrows point to NB84-positive cells).

FIG. 4A-4E, according to some aspects of the invention, demonstrate SKPsinfected with GFP adenovirus. (4A) SKPs spheres were dissociated, platedon laminin/lysine-coated slides, and infected with AdGFP. Virtually allcells in the culture expressed GFP. (4B-4E) SKPs (expressing YFP)migrate when transplanted in ovo into the developing chick neural crest.(4B) Picture of a single YFP-labeled SKP. (4C) YFP-labeled cells in theDRG. (4D, 4E) SKPs migrate into peripheral neural crest targets anddifferentiate into glia. (4D) SKPs that have migrated into thesympathetic ganglia (ganglia labeled with βIII-tubulin) (NT). (4E) SKPsmigrating to the DRG label with the Schwann cell markers 100β.

FIG. 5A-5F, according to some aspects of the invention, demonstrates thephenotype of cultured primary NB tumors of different disease phenotypesgrown in serumfree culture conditions containing FGF and EGF.Sphere-like clusters formed in the majority of tumor phenotypesfollowing acute dissociation. Following passaging, high-grade tumorspheres from tumors (5A) and bone marrow aspirates (5B) were capable ofreforming and growing as spheres, with the exception of a singlehigh-grade tumor sample which acquired adherent growth characteristicsupon passaging (5C). Tumors with good prognosis including ganglioneuromatumors, a benign cousin of NB (5D), and low-grade NB tumors (5E)acquired adherent growth characteristics upon passaging. Scalerepresents 100 μm. (5F) Undifferentiated primary tumor spheres from allNB phenotypes were immunostained for characteristic clinical markers ofNB (NB84 and TH) and the characteristic SKPs stem cell markersfibronectin and nestin. Scale represents 50 μm.

FIG. 6A-6D, according to some aspects of the invention, illustrates thathigh-grade NB tumor sphere cells exhibited increased self-renewalcapacity and could be passaged multiple times when compared to low-gradetumors and SKPs (negative) controls. Serial dilutions of single cellsfrom tumor spheres were plated into semi-solid methylcellulose and thepercentage of single cells capable of forming a secondary sphere wascalculated. This process was repeated until the sphere-forming cellswere depleted. (6A) Average number of passages until self-renewalability was depleted (+SEM) for ganglioneuroma tumors and low-grade NBtumor spheres (low) and high-grade NB tumor spheres (high). The numberof spheres that formed was proportional to the number of cells platedand did not alter with passaging (6B) and growth curves indicatedlogarithmic growth of cultured primary tumors with time (+SEM) (6C).High-grade tumor spheres (patient 5) and adherent cells (patient 12)retained their immunophenotype for NB84 and TH with passaging (6D),scale represents 50 μm.

FIG. 7A-7C, according to some aspects of the invention, demonstratesthat high-grade NB tumor sphere cells exhibited limited differentiationpotential when compared to tumor spheres from low-grade NB tumors. Tumorspheres were differentiated under neurogenic conditions andimmunostained with a variety of neuronal markers. Neuronal networksformed from both low and high-grade tumor spheres and retainedexpression of the clinical NB markers NB84 and TH (7A). Spheres fromlow-grade tumors differentiated into large nestin positive orβIII-tubulin positive neuronal networks (B, upper panel). High-grade NBtumor samples formed nestin positive and βIII-tubulin positive neuronsand fewer neuronal networks (7B, lower panel). Scale represents 100 μm.Similar proportions of tumor spheres from both low and high-grade NBtumors formed single neurons upon differentiation whereas only low-gradeNB tumor spheres were able to form complex neuronal networks upondifferentiation (+SEM) (7C).

FIG. 8A-8E, according to some aspects of the invention, demonstratesthat tumor spheres from a high-grade NB bone marrow aspirate (patient 5)were dissociated and injected orthotopically into the adrenal fat padsof SCID/Beige mice. (8A) H&E staining showing as few as 100 unselectedcells injected orthotopically formed micro-tumors 3 weeks afterinjection, whereas 10⁴ cells formed large tumor masses in the same timeperiod. Tumors stained positive immunohistochemically for the clinicalNB markers NB84 and TH and for the neural progenitor cell marker nestin(arrow heads). (8B) H&E staining showing cells that had metastasized tothe liver (left) and invaded the surrounding kidney (right). The time tomorbidity decreased with increasing number of cells injected (8C) andthe proportion of animals exhibiting tumors, either microscopic or gross(8D), and distant metastases (8E) increased with increasing cell dose.Error bars represent SEM.

FIG. 9A-9E, according to some aspects of the invention, provides a flowcytometry analysis of high-grade NB TICs. Cells were negative for thebrain TIC marker CD133/1 (9A) and highly positive for the clinical NBmarker NB84 (A) and CD271/p75 (9B). A small fraction of TICs stainedpositive for the metastatic marker CD24⁺ (9B) in two independenthigh-grade NB tumor sphere populations (patients 5 and 14) and was notexpressed by ganglioneuroma (patient 4) tumor spheres. Small numbers ofbrightly positive CD24⁺ cells were observed in NB tumor spheres byimmunocytochemistry (9C). Similarly, a small fraction of TICs stainedpositive for the progenitor cell marker CD34⁺ (9D) in two independenthigh-grade NB tumor sphere populations (patients 5 and 14) and was notexpressed by ganglioneuroma (patient 4) tumor spheres. Small numbers ofbrightly positive CD34⁺ cells were observed in NB tumor spheres byimmunocytochemistry (9E). The CD24⁺/CD34⁺ cell fraction from high-gradeNB tumor spheres reduced the time to morbidity when injectedorthotopically into immuno-compromised mice (+SEM) (9F), enriching thetumor-forming potential of these cells.

FIG. 10, according to one embodiment of the method, presents adiagrammatic flow chart demonstrating the design of the high throughput,dual-cell (Normal or Tumor cells) screening assay employed in theselection of candidate test compounds that target NB TICs. Normal or TICspheres are dissociated; 3,000 single cells/well are plated in 96 wellplates; candidate test compound is added; cell proliferation assayed byAlamar Blue signal. Blue/nonfluorescent compound is converted to ared/fluorescent compound under reducing conditions such as thoseproduced by live cells. The magnitude of the fluorescent signal isproportional to the metabolic activity of the cell sample.

FIG. 11, according to one embodiment of the invention, presents theresults from a study wherein FS90 (normal human SKPs, passage 3) cellswere treated with the LOPAC™ library of chemical compounds. alamarBlue®(a cell viability and proliferation indicator)_was added after 30 hoursand fluorescence intensity read after an additional 24 hours. The hitcutoff is indicated in the graph by the thick line across the graph atthe Y axis value of about 69.00% Control alamarBlue® Signal (whichcorresponds to 3 standard deviations from the mean of all test samples).Nine compounds whose alamarBlue® signals fall below this line wereidentified as primary hits in this study. (X axis presents the CompoundID number (n=80×8 plates); Y axis presents the % Control alamarBlue®Signal).

FIG. 12A-12C, according to one embodiment of the invention, presents thestudy results from primary screens of the chemical libraries examined.12A presents the results of the primary screen in Venn diagram form. TheVenn diagrams depict the primary hits from each library. Compounds inthe gray-bordered circles (left circle) affected the tumor-initiatingcells, while compounds in the black-bordered circles (right circle)affected normal cells. Compounds that affected both cell types lie inthe overlap region. Note that there is some compound redundancy betweenthe libraries. 12B presents the confirmed primary hits in Venn diagramform. Primary hits were retested against NB12, FS90 and FS105 (normalhuman SKPs). 87% of the primary hits were confirmed in this step,yielding 54 unique compounds that target tumor-initiating cells, 4unique compounds that target normal cells, and 46 compounds that haveactivity against both normal and tumor cells (overlap region). 12Cpresents in a pie-format the classification of primary hits by mechanismof action. (Solid light gray area=DNA damaging agents/cell cycleinhibitors; Solid dark gray area=Na⁺/K⁺ ATPase inhibitors; Diagonalstriped area=Neuronal receptor effectors; Vertical striped area=Other;Solid white area=Metabolic inhibitor; Checkerboard area=Neuronal channeleffectors; Dotted area=Specific protein effectors).

FIG. 13A-13E, according to one embodiment of the invention, presents theIC₅₀ values that were determined for the 64 selected candidatecompounds. Compounds were chosen for further testing based ondifferential cell type selectivity, mechanism of action, andpharmacological interest. Tumor-initiating cells and normal cells weretreated with 10 serial dilutions of compounds (5 μM to 9 nM).Representative graphs are shown in FIG. 13A (Complete Response), 13B(Partial Response), and 13C (Threshold Effect). Compounds that affectedthe tumor-initiating cells at a much lower dose than normal cells (13Dgraph, left) or compounds that had a greater effect on thetumor-initiating cells than normal cells (13E, right graph), wereselected for secondary in vitro screens in addition to those compoundsthat only affected tumor-initiating cells. (FS90=normal cells;NB12=tumor-initiating cells).

FIG. 14A-14C, according to one embodiment of the invention, presents theresults from secondary screens of the candidate compounds. Compounds ofinterest are being tested against additional normal primary cultures(FS89, FS105), a stage 1V neuroblastoma primary culture (NB25), and aneuroblastoma cell line (KCNR) using a sphere formation assay. 14Apresents a flow diagram of the secondary in vitro screen. The candidatecompound is added at 0 days and at 3 days. Spheres are counted at 7days. FIG. 14B presents a dose response curve of various cell lines(FS89, FS90, FS105, NB12, NB25 and KCNR) to amsacrine. FIG. 14C presentsa dose response curve of various cell lines (FS89, FS90, FS105, NB12,NB25 and KCNR) to MG624.

FIG. 15A-15FF, according to one embodiment of the invention, presentsIC₅₀ values for 32 selected compounds from the LOPAC™ and PRESTWICKCHEMICAL_LIBRARY® collections. Tumor-initiating cells (NB12) and normalcells (FS90) were treated with 10 serial dilutions of compounds rangingfrom 5 μM to 9 nM. Cell survival/growth was assayed using alamarBlue®and the percentage of control alamarBlue® signal was plotted versus log[compound] nM. IC50 values for NB12 are given above each plot.

FIG. 16A-16FF, according to one embodiment of the invention, representsIC₅₀ values determined for 32 selected compounds from the LOPAC™,PRESTWICK CHEMICAL LIBRARY®, and SPECTRUM™ collections. Tumor-initiatingcells (NB12) and normal cells (FS90) were treated with 10 serialdilutions of compounds ranging from 5 μM to 9 nM. Cell survival/growthwas assayed using alamarBlue® and the percentage of control alamarBlue®signal was plotted versus log [compound] nM (FS90 in dashed line, NB12in bolded line). IC_(so) values for NB12 and FS90 are given beside eachplot.

DETAILED DESCRIPTION

It is advantageous to define several terms before describing theinvention. It should be appreciated that the following definitions areused throughout this application.

DEFINITIONS

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For the purposes of the present invention, “a”, “an” and “the” includereference to the plural unless the context as herein presented clearlyindicates other wise.

For purposes of the present invention, the term “active agent” isdefined as a chemical entity, group of chemical entities or compoundthat is capable of providing an affect on NB TICs or NB cells in vitroor in vivo. The affect of the active agent may be a reduction incytotoxicity relative to the level of cytotoxicity demonstrated in theabsence of the active agent under similar conditions, or a cytostaticaffect on NB or on NB TICs that results in a reduced rate of NB or NBTIC proliferation and/or growth, or a reduction of the rate oroccurrence of differentiation into NB cell types, precursors, or anyother cell type that is related to the progression of a NB pathology, orto an increase in the inducement of the differentiation of NB TICs intocell types (for example, neurons) that no longer proliferate (forexample, retinoic acid is a common differentiation therapy forneuroblastoma that is used as an adjunct therapy after removal of atumor, differentiation therapy).

For purposes of the present invention, the term, “effective amount” isdefined as an amount of a compound that will inhibit and/or reduce NBTIC survival, proliferation, or that will promote the differentiation ofNB TICs into benign cell types.

For purposes of the present invention, the term “enriched” is defined ascontaining a higher percentage of a particular cell type, such as acancer stem cell, than is typically present in a native, non-enrichedpreparation. For example, as used in the definition of the presentinvention, an “enriched” preparation may be defined as a function of thepercentage of tumor initiating cells capable of giving rise to tumorcells in a preparation. An enriched preparation of neuroblastoma tumorinitiating cells comprises a greater percentage of neuroblastoma tumorinitiating cells capable of giving rise to secondary neuroblastomaspheres compared to a non-enriched preparation. In some embodiments, anenriched preparation of neuroblastoma tumor cells may be described ascomprising about 2% or greater, or about 3% to about 18% of the totalcell population contained in a preparation. By way of comparison, anon-enriched preparation of neuroblastoma cells would include only about0.2% to about 2.0% or less neuroblastoma tumor cells that are capable ofgiving rise to a secondary neuroblastoma sphere. In some embodiments,the enriched preparations comprise a 100-fold, 200-fold, 500-fold,1.000-fold, or up to a 2.000-fold or 10.000-fold to 20.000-fold enrichedpreparation of neuroblastoma cells capable of giving rise to secondaryneuroblastoma spheres. Since 2×10⁶ cells from established NB cell linesare typically required to form tumors using the protocols describedherein, an approximately 20.000-fold enrichment in tumor inititiatingcells is provided.

For purposes of the present invention, the term “neuroblastoma tumorinitiating cell” (NB TIC) is defined as a cell that is capable of givingrise to NB or a tumor cell that is identifiable with a condition of NB,such as a tumor cell that may be identified to have particularidentifiable cell surface markers associated with NB (such as NB84,CD44, TrkA, GD2, CD24, CD34, p75NTR, and/or versican) and/or is withoutcell surface markers that are characteristic of tumor cells that are notfrom NB (such as CD133, TrkB, and/or CD31). For purposes of the presentinvention, the term “neuroblastoma tumor-initiating cell inhibitingactivity” is defined as an activity for affecting NB TIC survival,proliferation, or that promotes cell differentiation into benign celltypes.

EXAMPLES Example 1 Isolation and Characterization of Tumor InitiatingCells (TICs) in Neuroblastoma Tissue

The present example demonstrates the utility of the present inventionfor providing an enriched preparation of TICs, particularity NB TICs, ata high concentration.

TICs comprise a relatively rare cell population within tumors. Forexample, brain tumors contain 0.3% to 25% TICs, depending on tumor grade(13, 14). The present example demonstrates the utility of the presentinvention for providing a highly enriched preparation of specific TICsthat are derived from bone tissue, particularly bone tissue from apatient having been diagnosed with NB. The bone marrow employed toprovide these enriched preparations of NB TICs had metastasized.

Materials and Methods:

Neonatal adrenal gland and SCG were dissociated and cultured under SKPconditions.

Fourteen (14) neuroblastoma (NB) samples including 9 unfavorableprognosis (stages 3 and 4) and 5 favorable-prognosis tumors(ganglioneuroma or benign NB, and stages 1, 2, and 4S) were obtained.Samples were obtained from both tumor tissue and bone marrow metastasesthat were diagnosed cytomorphologically as clumps of neuroblasts.

NB TICs were isolated from bone marrow aspirates, since (a) they are ahallmark of the highest grade NB (20), (b) it is an accessible sourceobtained at serial time points before and after chemotherapy, (c) bonemarrow contains no resident NCPs (16), and (d) bone marrow from NBpatients is tumorigenic when injected into NOD/SCID mice.

The dissociated tumor or bone marrow cell clumps were cultured in humanSKP conditions. Human SKP conditions in a culture using uncoated flaskscontaining defined medium, EGF and FGF (16, 18).

Over one week, spheres of proliferating cells appeared, as seen in phaseillumination (See FIG. 1A). While many cells adhered to the plastic ordied, within 1-10 weeks, spheres of proliferating cells formed (FIG. 2A)which upon dissociation and passaging proliferated and formed newspheres. The majority of samples formed primary tumor spheres, all ofwhich expressed the NB markers NB84 and TH and the SKPs markersvimentin, versican, nestin and fibronectin (FIG. 2B-F). These tumorspheres were selected to provide the enriched preparation of NB TICs.

Example 2 Self-Renewal and Proliferation of NB Tumor Spheres

One of the characteristics of a TIC is that it self-renews over anextended period of time either in culture or in vivo. The presentexample demonstrates that the NB cancer stem cells, or NB TICs of theinvention, posses the ability to self-renew over a defined period oftime.

In the present example, a methylcellulose assay (colony sphere-formingability of single cells is assessed in methylcellulose), and alimiting-dilution assay (growth of isolated single cells is assessed)will be used to demonstrate the activity of the NB TICs identified inthe present invention. In self-renewing addition, the proliferative rateof the tumor spheres will also be determined by BrdU labeling.

Methylcellulose Assay:

The percentage of NB sphere-forming cells was assessed by plating in0.8% methylcellulose. This technique has previously been used for rodentSKPs (17).

In initial studies, 0.2% to 2.0% of the starting tumor cell populationformed spheres. A similar assessment of dissociated primary tumorspheres showed that up to 18% of these cells could form a secondarysphere, an enrichment of up to 100-fold in cells capable of growing ascolonies in semi-solid medium. In both cases, the number of spheresformed was proportional to the amount of cells plated (FIG. 3B).

Limiting Dilution Assay:

Four (4) of the tumor sphere samples have been passaged 3 or more times,with frequencies ranging from 0.05% to 18% for a stage 1/4S verses stage4 tumor, respectively. These data indicate that the tumor spheres canself-renew, a major criterion for TICs.

One of the tumor sphere samples derived from a bone marrow aspirate of astage 4 tumor from relapsed disease (NB12) has now been dissociated andpassaged 28 times in liquid culture over a 60 week period. In all cases,passaged and primary tumor spheres displayed the same phenotypicprofile. All of the tumor sphere samples will undergo self-renewalanalysis; (b) all self-renewal results will be confirmed using limitingdilutions assays, and (c) cell surface markers will be identified toprospectively-identify NB TICs by flow cytometry.

It is anticipated that tumor spheres from high stage tumors willself-renew for longer periods of time and at higher frequencies thanthose from more differentiated low stage and benign tumors. It is alsoanticipated that all of the tumor spheres will generate sympatheticneuroblasts at some frequency.

Example 3 NB Tumor Spheres differentiate into Sympathetic Neuroblastsand Schwann Cells

NB tumor spheres differentiated into sympathetic neuroblasts, a celltype diagnostic for NB. The present example establishes that the cellsisolated according to the present invention are TICs for NB, as they areshown herein to differentiate into sympathetic neuroblasts and Schwanncells, the hallmark NB cell type.

When differentiated in conditions used to generate sympathetic neuronsfrom SKPs, tumor spheres from three low stage and one high stage tumorgenerated morphologically complex cells that were positive for TH, andthe neuron-specific proteins βIII-tubulin and NFM (FIG. 3A). Moreover, asubpopulation of differentiated cells expressed the Schwann cell markers100β GFAP, and GalC. In contrast, spheres from two other stage 4 tumorsremained largely undifferentiated, and generated only a few cellsexpressing neuronal or glial proteins. These phenotypes and expressionpatterns are typical of low and high stage NB. Thus, the NB tumorspheres can regenerate cell types of the original tumor, fulfillinganother criterion for TICs.

Example 4 NB Tumor Spheres form Tumors In Vivo

Defining characteristics of TICs are that they (a) are highly enrichedfor tumor-forming ability relative to the initial tumor cell populationand (b) can recapitulate the phenotype of the original tumor in vivo(4). The present example demonstrates that as few as 100 NB tumor spherecells (the lowest number examined) isolated according to the presentinvention will form a NB tumor when injected subcutaneously in mice(FIG. 3C, 16, 17), as compared to the 10⁶ cells that are normallyrequired. Since 2×10⁶ cells from established NB cell lines are typicallyrequired to form tumors using this protocol (28), this represents anapproximately 20.000-fold enrichment in TICs.

The tumor spheres will be used to identify the oncogenic eventsresponsible for maintenance of the tumor phenotype.

Model 1:

To assess whether the NB tumor spheres could form tumors, ten primaryspheres (2,500 cells) isolated from a needle biopsy of a tumor (sampledesignated NB05b) obtained from a patient newly diagnosed with stage 4NB, were injected subcutaneously into the flanks of NOD/SCID mice, axenograft model of NB (50).

A large tumor arose that contained cells resembling immature neuroblastswith small refractile cell bodies and high nuclear to cytoplasmicratios, and that immunostained with the NB marker NB84 (FIG. 3C). Sincemost NB xenograft models with subcutaneous injection require 10⁵-10⁶cells to form visible tumors, this result indicates that the NB05 tumorspheres were highly enriched for tumorigenicity, consistent with theiridentity as TICs. Cells from primary spheres of a second stage 4 bonemarrow (NB12) also formed tumors in mice. Tumor formation will beassessed for all of the NB tumor sphere lines in an orthotopic model ofNB tumor formation. A small piece of in vivo primary tumors from bothNB05b and NB12 patients were used to re-implant and form secondarytumors in vivo.

Model 2—Orthotopic Model:

An orthotopic model of NB will be used to assess tumor formation by NBtumor sphere cells. In this model, injection of NB cells (between 1×10²and 1×10⁵) into the mouse adrenal fat pad (a common site of NB) resultsin primary tumor growth in the adrenal and distant metastasis to sitessuch as bone marrow (28). Moreover, these tumors closely resemble humanNB. Tumors in this model can be easily quantified 21 days afterinjection of NB cells. The initial analysis of the NB spheres isconducted by injecting dissociated tumor sphere cells from one stage 4(NB12) and one ganglioneuroma (NB10) NB.

The different cell populations will be compared for tumorigenicity byinjecting from 1×10² to 1×10⁵ dissociated cells into the adrenal fatpad. Cells to be injected include (a) unsorted tumor spheres fromdifferent NB stages, (b) FACS-sorted tumor cells that are both positiveand negative for tumor sphere markers, (c) cells that have been sortedfor tumor sphere markers and then expanded as tumor spheres, and (d)acutely dissociated total NB cells. Tumor masses will be assessed at1-24 weeks, time points based upon the study of Dirks with orthotopictransplants of brain TICs (14). Tumors will be assessed histologicallyfor morphology typical of NB, by immunohistochemistry for NB84, TH,vimentin, nestin, and βIII-tubulin (all markers for NB neuroblasts), andfor proliferation by immunostaining for Ki67. A portion of thesesecondary tumors will also be dissociated, the cells resorted by flowcytometry, and then either directly injected into mice to determinewhether they can be serially passaged (a characteristic of other TICs),or expanded in sphere cultures and characterized as for the initialtumor cell population.

These studies demonstrate the existence of a tumor-initiating stem cellin NB, and provides a system that may be used to characterize how thistumor arises and progresses.

Example 5 NB Tumor Spheres exhibit Abnormal Karyotypes

To confirm that the NB tumor spheres were derived from NB, metaphasespreads of multiple clones of tumor spheres from sample NB12 (one of thestage 4 samples that generated a tumor) were karyotyped.

Three populations of cells were identified, one 76% diploid and theothers tetraploid, with and without double minutes, karyotypes typicalof stage 4 NB.

A detailed assessment of clonal tumor sphere cells for DNA content,amplification of MYCN, and loss of heterozygosity (LOH) that oftenoccurs in high stage NB, particularly deletion of 1p, trisomy of 17q,and 11q LOH (1), will be undertaken.

The detailed genomic assessment will include an assessment of expandedclonal tumor spheres for DNA content, amplification of MYCN, and LOHthat often occurs in high stage NB and particularly 1p, 11q and 17q LOH(1). This will be accomplished on metaphase preparations of dissociatedprimary tumor spheres by GTG banding and by single nucleotidepolymorphism (SNP) analysis using high-density oligonucleotide arrays.SNP analysis will be particularly valuable for assessing loss ofheterozygosity that is common in high grade NB. Expansion of singleclonal spheres of human SKP cells has already been achieved, and similarstudies (18) have already been performed.

Example 6 Cell Surface Marker Expression

The present example shows that a subpopulation of cells from an originalNB tumor can reform tumors, and that these reformed tumor cells possessidentifiable cell surface markers associated with NB, including but notlimited to NB84, CD44, TrkA, GD2, CD24, CD34, p75NTR, and/or versican.

Flow cytometry was used to identify cell surface markers for NB tumorspheres. These markers will be used to prospectively isolate potentialTICs directly from the tumor. Antibodies known to recognize cell-surfaceproteins that are (a) most highly expressed in high grade NB tumors,including CD44, TrkB, and GD2 [the latter two specific to high grade orrelapsed NB, (1,25)], (b) preferentially expressed on low-grade NB andhuman SKPs, including p75NTR (26,27), (c) are expressed on SKPs, butthat have not been tested in NB, including versican, will be used, and(d) other markers of interest in neural crest development and cancersincluding NB, such as CD₂₀, CD56 and CD29 (57-59).

Among the cell surface markers that are not expressed on SKPs or highgrade NB and that will be tested for negative selection are CD106(mesenchymal stem cell marker), TrkA, and CD31 (endothelial precursormarker). This strategy is anticipated to be highly selective, as NBtumor spheres have already been shown to express NB84 and versican, anda similar strategy has been used by flow cytometry to prospectivelyisolate SKPs from rodent dermis (26).

The “cell surface signature” identified according to this procedure forNB sphere-forming cells will be used to sort tumor cells intopopulations that are either positive or negative for these markers. Themarker presence or absence will be confirmed using RT-PCR andimmunocytochemistry. The positive and negative populations will then beassessed for (a) their capacity to self-renew, using the sphere assay,(b) karyotypic abnormalities characteristic of NB, and (c) their abilityto re-form tumors in vivo. A subpopulation of NB TICs will be isolatedthat will express markers of SKPs and NB, and that will subsequentlyself-renew as spheres and reform tumors, while the negative populationwill neither self-renew nor form tumors.

The tumor-initiating ability of high-grade tumor spheres is highlyenriched in the CD24⁺/CD34⁺ fraction of high-grade NB tumor spheres(FIG. 20). CD24 was chosen as a putative marker of NB TICs because itwas reported to be expressed on renal cell carcinomas, small cell lungtumors, breast cancer TICs as well as NB (64-65). CD34 was chosen as aputative marker of NB TICs because it had been reported that patientsrelapsed with NB following CD34⁺ peripheral blood stem celltransplantation (62). The CD24⁺/CD34⁺ Fraction of NB TICs was found tobe highly enriched for tumor-initiation in an orthotopic model of NB.

Example 7 Low Stage NB, High Stage NB, Bone Marrow Metastasis andRelapsed Tumor Characterization of TIC Populations

Neuroblastoma TICs from different grades of NB, obtained from tumors andmetastases, and before and after relapse, will be used to identifymolecular differences between these cells and others, and to determinehow NB initiates and progresses, and why some NB are benign and othersare fatal. Identifying these differences will be used to definetherapies specific for individual patient NB.

Marker and gene expression differences will be assessed on differentpopulations of tumor spheres by (a) comparative immunocytochemistry, (b)RT-PCR, and (c) expression profiling using NB-specific cDNA microarray.For immunocytochemistry, NB tumor spheres will be analyzed for the NBmarkers NB84 and TH, the SKP markers versican, vimentin, nestin, andfibronectin, the neuronal markers NFM and βIII-tubulin, the Schwann cellmarkers s100β, GFAP and CNPase, and the cell surface antigens defined inflow cytometry studies.

For RT-PCR, expression of genes will be assessed that are (a) enrichedin high-grade NB (as determined by cDNA microarray), (b) that regulateneural crest development such as hAsh, hTwist, and Id2 (29), (c) theunfavorable prognosis markers TrkB, MYCN, and mutant Phox2b, and (d) thefavorable prognosis marker TrkA. Western blot analysis will be performedwhen antibodies are available that recognize the human proteins, such asfor TrkA and TrkB. These data will be used to choose a limited number ofsamples for expression profiling, which will be accomplished using aNB-specific cDNA microarray reported (30). This analysis is expected topredict unfavorable and favorable NB at a very high accuracy.

Candidates will be selected based on further analysis of the data toidentify candidate genes to test (30). Total RNA from 1×10⁷ TICs, anumber that can be obtained with at least two of the NB tumor spherecultures. Genes preferentially expressed in TICs from high or low-gradeNB and from bone marrow or following relapse, and that have beenimplicated in the control of cell growth, survival, metastasis ortumorigenicity will be assessed for their role in NB.

Example 8 Molecular events in Transformation of Neural crest precursorsinto NB TICs, and Suppression of Tumorigenic Properties of NB TICs

To identify the molecular events involved in the transformation ofneural crest precursors into NB TICs, human NCPs and NB TICs will begenetically manipulated with oncogenes or siRNAs to potential tumorsuppressors of NB, and re-implant these into (i) the adrenal fat pad inmouse where most NB tumors arise, and (ii) the chick neural crestmigratory stream, a system where NCPs differentiate into neural crestprogeny, thereby permitting the definition of the developmental stage atwhich potential oncogenes cause tumor formation.

Among the genes to be assessed will be the unfavorable prognosis NBmarkers MYCN, Id2, h-Twist, mutant Phox2b, and ΔNp73, and the favorableprognosis markers TrkA and overexpressed Phox2b. Genes will also beassessed that are preferentially expressed or suppressed in TICs fromdifferent stages of NB.

Example 9 Molecular Events in the Appearance and Progression of NB

The molecular events that regulate the appearance and progression of NBare relatively uncharacterized. Of the unfavorable prognosis markers,only MYCN has been shown to induce NB when over expressed in atransgenic model that targets sympathoadrenal precursors and theirdifferentiated progeny (31). However, MYCN-induced NB in mice arisesmuch later than human NB, and rarely metastasizes, suggesting that otheroncogenic events are required to reproduce the full NB phenotype, orthat events that induce NB differ in mouse and human.

The proliferative and tumor-inducing potential of several genesimplicated in NB will be assessed, in the three cell types human NBtumor spheres, human SKPs, and rodent adrenal gland precursors. Thegenes that will be initially tested are MYCN, Id2, h-Twist, ΔNp73,Phox2b, TrkB, and TrkA. The rationale for choosing these genes is asfollows.

(i) MYCN is the best-known poor prognosis marker in NB, correlating wellwith rapid tumor progression, poor outcome, and treatment failure (1).It is amplified and over expressed in 22% of high stage NB, and itsinhibition is required for the cell cycle arrest of sensory precursorcells (1,32). MycN may function as a proliferative protein, or suppressthe expression of genes important for cell cycle arrest anddifferentiation, such as TrkA. (ii) Expression of the Id2 inhibitoryhelix-loop-helix (HLH) protein strongly correlated in one study withpoor outcome in NB (33) and MycN-mediated cell cycle progressionrequires the Id2-induced suppression of Rb activity (33). (iii) h-Twistis a bHLH transcription factor that is expressed primarily in MYCNamplified NB (34). It is required in at least one NB cell line tooverride the apoptotic activity of MYCN by suppressing ARF and p53activity (34). MYCN is likely to be tumorigenic only in cells overexpressing h-Twist, mutated p. 53 (rare in NB), or suppressors of p53activity such as ΔNp73. (iv) Phox2b is a homeobox domain transcriptionfactor that in mice is required for differentiation of noradrenergicneurons, and that together with Mash1, drives progenitors to becomepost-mitotic sympathetic neurons (35, 36). Frameshift germline Phox2bmutations (R100L) have been reported in a subset of familial NB, whileover expression of wild type Phox2b correlates with favorable prognosis(3).

Mutations in Phox2b may therefore block the differentiation ofsympathoadrenal precursors and contribute to NB, possibly by dimerizingwith and inhibiting wild-type Phox2b. (v) ΔNp73, a p53 family member, isa major survival protein in the sympathoadrenal lineage (37). ΔNp73expression strongly correlates with poor outcome in NB, and is detectedin cells lines with amplified MYCN (38). Since this protein can ablatep53 activity (37), it may collaborate with MycN (the protein) to induceproliferation, and/or promote the survival of sympathoadrenal precursorscontaining unstable genomes. (vi) TrkA is a poor-prognosis NB markerthat has been shown to induce survival, migration and invasion, andresistance to chemotherapeutic agents when expressed in NB cell lines(1, 39, 40). It was also observed that TrkB activation induces NBneuronal differentiation (41). However, TrkB-expressing NB cellscontinue to proliferate as they differentiate, which is similar to theneuroblast phenotype of NB tumors. Unfavorable tumors also express BDNF,the TrkB ligand (1). (vii) The expression of the TrkA Nerve GrowthFactor (NGF) receptor is highly correlated with favorable NB outcome,lower stage, and absence of MYCN amplification (1). TrkA inducesneuronal differentiation, suppression of MYCN levels, cell cycle arrest,and apoptosis in NB cell lines, depending upon its expression level (1,42, 43). TrkA may function by promoting the terminal differentiation anddeath of inappropriately cycling neuroblasts.

The above data suggests that NB proto-oncogenes can be grouped intothose that induce or maintain the proliferation of progenitors and blockdifferentiation of their post-mitotic progeny such as MYCN, Id2, andmutant Phox2b, and those that prevent p53 function or that are potentsurvival proteins such as h-Twist, TrkB, and 4Np73. The expression ofany pair of pro-proliferation and survival proteins will transform SKPsor adrenal precursor cells to a NB fate. Likewise, over expression ofTrkA or wild type Phox2b, or suppression of the expression of the aboveproteins via siRNA, will inhibit the proliferation and tumorigenesis ofNB TICs and induce their differentiation.

The functional importance of genes preferentially expressed in TICs fromhigh or low-grade NB, from bone marrow, and following relapse, will alsobe assessed.

Data, Model Systems

Three cell systems; human SKPs as a human NCP, mouse neonatal adrenalprecursor cells, and human NB TICs will be used. The sorted, expanded NBcells for these studies will be used. The unsorted NB tumor spheres mayalso be used. These genes will be introduced into these cells usingadenovirus or lentivirus. Several hundred recombinant adenovirus vectorsencoding epitope-tagged genes and GFP have been constructed, and will beused for the functional analysis of proteins in neurons and progenitors,including MYCN, TrkA, TrkB, Id2, and ΔNp73 (37, 43-46). Moreover, thesegenes have been for up to 7 days in SKPs using recombinant adenovirus(FIG. 4A). However, since gene function will be assessed inproliferating cells in vivo, the lentivirus vectors will also be used,which efficiently integrate into genomes and infect precursor cells atefficiencies of 80-90% (47, 48).

Example 10 In Vivo Systems to Assess Tumorigenesis and Differentiation

The present example is presented to demonstrate the utility of thepresent invention for providing an in vivo model for mammalian neuralcrest differentiation. The methods thus posses the major advantage thatthe transplanted, transformed precursors will undergo the samedifferentiation steps that they would during human development, therebypotentially unmasking effects that are differentiation-stage specific.Aberrant growths arising from transformed CNS neural precursors withinthe chick spinal cord in ovo have previously been observed, even furtherestablishing the feasibility of studying tumorigenesis in this system.

Genetically manipulated cells will be implanted into (i) the adrenal fatpad in mouse, and (ii) the embryonic chick neural crest migratory streamin ovo. Single SKP spheres transplanted into the latter system migrateinto peripheral neural crest targets, including the spinal nerve, DRG,and sympathetic ganglia (the latter a site for NB), and differentiateinto neural crest progeny (FIG. 4B-E, 17).

Example 11 Candidate NB Oncogenes and SKPs, Adrenal Precursor Cells,and/or Cells from Low-Stage NB and Proliferation Activity In Vitro, andin the Formation of Tumors

NB neuroblasts and the NB tumor spheres continue to proliferate even asthey express sympathetic neuron markers. SKPs, adrenal precursors, andcells from low-stage NB, will be used to express the proliferative NBoncogenes in combination with the pro-survival oncogenes that have beendescribed above, and proliferation and differentiation of these cellswill be monitored (a) under the sphere expansion conditions describedherein, in FGF and EGF, and (b) under normal neural differentiationconditions as described herein, in the absence of mitogens. Note thatSKPs will not form tumors when implanted into chick (17) or mice (49).For these studies, one proliferative and one prosurvival protein will beused per study (see FIG. 5).

Cells will be co-transduced with GFP as a marker, and proliferation willbe monitored by BrdU labeling and Ki67 immunocytochemistry, anddifferentiation into sympathetic neuroblasts or neurons byimmunocytochemistry for nestin, vimentin, TH, βIII-tubulin, and NFM.Expression of the virally-expressed proteins will be confirmed byimmunocytochemistry and Western blotting, as done for MYCN, Id2, andTrkB (43, 45,46). For TrkB, cells will be treated with BDNF, and TrkBtyrosine phosphorylation assessed (41). One or more combinations ofthese potential oncogenes, for example MYCN and 473, is anticipated tocause the appearance of proliferating cells with characteristics ofsympathetic neurons i.e. NB neuroblasts. In contrast, control SKPs andadrenal precursors will differentiate into post-mitotic sympatheticneurons, as previously shown (18, FIG. 1C).

Having established which single/combinations of proto-oncogenes perturbproliferation or differentiation in culture, it will then be determinedif these perturbations also cause human SKPs, rodent adrenal precursors,or cells from low-stage NB to form tumors in vivo. Human SKPs aregenetically and phenotypically stable for over a year in culture (18),and neither rodent nor human SKPs form tumors in chick, mice or rats(17). However, it is not yet known whether the low-stage NB tumor spherecells will form tumors; if they do, potential enhancement of tumorgrowth will be assessed. The genetic manipulations in these studies willbe similar to those described for culture analysis, except thatlentiviral transduction will be used.

For transplantation into the orthotopic adrenal fat pad model, 1×10² to1×10⁴ transformed, GFP-tagged cells will be implanted and tumor growthwill be quantified for engraftment rate at 1-24 weeks post injection.Tumors will be assessed by histology for neuroblasts, andimmunocytochemically for the NB markers NB84 and TH, the proliferationmarker Ki67, and for the epitope tag on the expressed transducedprotein(s) as described (50). For transplantation into the embryonicchick, the transduced cells will be grown as spheres, and implanted intothe chick neural crest migratory stream (FIG. 4B-E, 17). Cell migrationto peripheral neural crest targets will be confirmed by the presence ofGFP-positive cells (FIG. 4B-E), and tumor masses identified as describedherein.

Example 12 Overexpression of TrkA or Phox2b, NB TICs Dividing Activity,Differentiation, Apoptosis and Tumorigenicity

A converse study will be conducted to (i); express TrkA or Phox2b indissociated NB tumor sphere cells from stage 4 tumors, and observed foractivity to differentiate or die and/or suppresses tumorigenicity. Thesestudies will be performed in culture, and will transduce the cells andassess cellular proliferation and differentiation as described in (i)and apoptosis by TUNEL.

NGF will be added to cells expressing TrkA. From the gene expressionarray analysis, it will be determined which NB TICs express MYCN, Id2,h-Twist, mutant Phox2b, TrkB or ΔNp73. The levels of these putativeproto-oncogenes will be selectively suppressed using shRNA viral vectorsor siRNA in those cells. The cells will be examined to determine if thisinhibits their proliferation and/or promotes their differentiation ordeath. Similar approaches have previously been used to manipulateprimary CNS precursors in culture and in vivo (51).

To determine if these same manipulations inhibit NB tumor spheretumorigenicity, the manipulated cells will be implanted into the mouseadrenal fat pad and the chick neural crest migratory stream, andtumorigenicity as described for (i) above will be assessed. One or moreof these approaches will be examined to determine if they suppress thetransformed phenotype of NB TICs. However, it is possible that thehigher-grade NB TICs may carry so many genetic perturbations that singlemanipulations will be insufficient to reverse their phenotype. If thisproves to be the case, then similar studies with lower-grade NB tumorspheres which carry fewer genetic perturbations will be conducted.

Isolation of NB TICs and of several types of NCPs will permit thecharacterization of molecular events regulating the transformation andprogression of NB, and whether there are molecular and phenotypicdifferences in cells from different stages of NB.

Example 13 Screening Method to Detect Alterations in CellViability/Proliferation

The present example provides a description of the screening method useto identify the chemical entities capable of affecting NB cells reportedin the present series of studies.

Malignant NB is the most common extra-cranial solid tumor in children.Survival of patients older than 1 year remains less than 30% withconventional therapies. Candidate NB TICs were isolated, and it washypothesized that TICs are related to SKPs. Both SKPs and TICs originatefrom the neural crest, express similar neural crest markers, anddifferentiate in vitro into similar cell types. The availability of twoneural crest stem cell sources, one from the NB tumor and the other fromthe skin of the same patient, affords us a unique opportunity fortherapeutic target discovery.

Study 1 Screen: Materials and Methods:

To identify compounds that suppress the growth and survival of NB TICsand not nontransformed normal cells (SKPs), a cell-based assay wasestablished and used in which NB TICs from a multiple relapse NB patient(NB12, passage 6-17) and normal SKPs (FS90, passage 2-5) were tested inparallel to detect specific alterations of cell viability/proliferation.For each cell type, cells were passaged 5 days prior to screening. Threethousand (3000) cells in 100 μL of SKPs growth media (B27, FGF, EGF,P/S, FUNGIZONE™ (antifungal agent) in DMEM:F12 with 50% hFS conditionedmedia) were robotically plated in individual wells of uncoated 96 wellplates and treated with test compound for 30 hours, prior to a 24 hourincubation in the presence of alamarBlue® and subsequent fluorometricreading. Under these conditions, the alamarBlue® signal displayed alinear response with time, background was minimal, and the dynamic rangesatisfactory (i.e., the alamarBlue® reading at 0 hours vs. 24 hourswas >10 fold different).

The robustness of the screen was initially evaluated by using acollection of 1280 bioactive compounds (LOPAC™ library, Sigma). For bothnormal SKPs and NB TICs, variability of signals was low, with CV valuesranging between 3.5-4.5% across the plates, and the dimensionless,statistical parameters Z′ and Z factors were >0.5, suggesting anexcellent assay quality. “Hits” were defined as the compounds whosesignals were shifted away by at least 3× standard deviations (99.73%confidence interval) from the mean of the general sample population.

Results:

The screen of the LOPAC™ library at 5 μM yielded 13 “hits” which werefound to affect both normal and NB cells. We also identified 18compounds that selectively target NB cells. Four compounds selectivelytargeted normal cells.

TABLE 2 13 compounds that affect both normal and NB cells: Ancitabinehydrochloride Brefeldin A from Penicillium brefeldianum Calmidazoliumchloride CGP-74514A hydrochloride Dihydroouabain Diphenyleneiodoniumchloride Emetine dihydrochloride hydrate Idarubicin Mitoxantrone OuabainQuinacrine dihydrochloride Ammonium pyrrolidinedithiocarbamateSanguinarine chloride

TABLE 3 18 compounds that selectively target NB cells. Loratadine MG 624Melphalan Podophyllotoxin Ro 25-6981 hydrochloride RotenoneDL-Stearoylcarnitine chloride Taxol Vincristine sulfate Vinblastinesulfate salt Chelerythrine chloride ColchicineCytosine-1-beta-D-arabinofuranoside hydrochloride Dequalinium dichloride(S)-(+)-Camptothecin Dequalinium analog, C-14 linker2,3-Dimethoxy-1,4-naphthoquinone Etoposide

TABLE 4 4 compounds selectively target normal cells:8-Methoxymethyl-3-isobutyl-1-methylxanthine Oligomycin A SphingosineThapsigargin

Study 2 Screen: Materials and Methods

The PRESTWICK CHEMICAL LIBRARY® was screened at 5 μM using FS90 and NB12and at 1 μM using NB12 only due to the high number of “hits” at 5 μM.This screen identified 9 compounds that selectively target NB12 and 15compounds that affect both NB12 and FS90.

Results:

TABLE 5 9 compounds that selectively target NB12: Azaguanine-8Paclitaxel Camptothecine (S.+) Colchicine Etoposide Doxorubicinhydrochloride Lanatoside C Podophyllotoxin Proscillaridin A

TABLE 6 15 compounds that affect both NB12 and FS90: DisulfiramMitoxantrone dihydrochloride Anisomycin Cephaeline dihydrochlorideheptahydrate Digitoxigenin Digoxin Strophantine octahydrate Puromycindihydrochloride Daunorubicin hydrochloride Emetine dihydrochlorideMethyl benzethonium chloride Strophanthidin Cycloheximide Thonzoniumbromide Sanguinarine

Study 3 Screen: Methods:

The results from the LOPAC™ and PRESTWICK CHEMICAL LIBRARY® screens wereconfirmed using FS90, FS105, and NB12. Thirty-six (36) compounds wereconfirmed that specifically affect NB12 and 29 compounds that affectFS90/105 and NB12. Thirty-two (32) compounds were selected for IC₅₀determinations using FS90, FS105, and NB12. IC₅₀ for the remainingcompounds of interest will be tested at a later date (in combinationwith hits from additional libraries).

Results:

TABLE 7 36 compounds that specifically affect NB12 (S)-(+)-Camptothecin2.3-Dimethoxy-1.4-naphthoquinone Ancitabine hydrochloride Antimycin AAzaguanine-8 Benzethonium chloride Camptothecine (S.+) Chelerythrinechloride Ciclopirox ethanolamine Clofazimine Colchicine ColchicineCycloheximide Cytosine-1-beta-D-arabinofuranoside hydrochlorideDequalinium analog. C-14 linker Dequalinium dichloride Dequaliniumdichloride Digoxigenin Diphenyleneiodonium chloride DL-Stearoylcarnitinechloride Doxorubicin hydrochloride Etoposide Etoposide MG 624Mycophenolic acid Paclitaxel Parthenolide PodophyllotoxinPodophyllotoxin Primaquine diphosphate Quinacrine dihydrochlorideQuinacrine dihydrochloride dihydrate Scoulerine Taxol Vinblastinesulfate salt Vincristine sulfate

TABLE 8 29 compounds that affect FS90/105 and NB12 Alexidinedihydrochloride Ammonium pyrrolidinedithiocarbamate Amodiaquindihydrochloride dihydrate Anisomycin Brefeldin A from Penicilliumbrefeldianum Calmidazolium chloride Cephaeline dihydrochlorideheptahydrate CGP-74514A hydrochloride Daunorubicin hydrochlorideDigitoxigenin Digoxin Dihydroouabain Disulfiram Emetine dihydrochlorideEmetine dihydrochloride hydrate Idarubicin Lanatoside C Methylbenzethonium chloride Mitoxantrone Mitoxantrone dihydrochloride OuabainProscillaridin A Puromycin dihydrochloride Sanguinarine Sanguinarinechloride Strophanthidin Strophantine octahydrate Terfenadine Thonzoniumbromide

TABLE 9 32 compounds selected for IC50 determinations using FS90, FS105,and NB12: (S)-(+)-Camptothecin Ammonium pyrrolidinedithiocarbamateAmodiaquin dihydrochloride dihydrate Antimycin A Avermectin B1Azaguanine-8 Chelerythrine chloride Clofazimine Colchicine Dequaliniumanalog, C-14 linker Dequalinium dichloride (LOPAC compound) Dequaliniumdichloride (Prestwick compound) Digoxin DihydroouabainDiphenyleneiodonium chloride DL-Stearoylcarnitine chloride EtoposideIdarubicin Loratadine MG 624 Myophenolic Acid Paclitaxel ParthenolidePodophyllotoxin Primaquine diphosphate Quinacrine dihydrochlorideSanguinarine chloride Scoulerine Strophanthidin Terfenadine Vinblastinesulfate salt Vincristine sulfate

Study 4—Screen Results at 5 μM:

The SPECTRUM™ collection was screened using the same protocol. At 5 μM,the initial screen identified 35 hits that affect NB12 and FS90, no hitsthat specifically target FS90, and 41 hits that specifically targetNB12. The screen was repeated at 5 μM and 104 using NB12 and FS90 toconfirm these hits and identified 34 hits that affect NB12 and FS90, nohits that specifically target FS90, and 33 hits that specifically targetNB12. Following the SPECTRUM™ confirmatory screen, 1050 determinationsfor an additional 32 compounds were performed.

TABLE 10 34 hits that affect NB12 and FS90: 3-METHYLORSELLINIC ACID5alpha-CHOLESTAN-3beta-OL-6-ONE 5-AZACYTIDINE AKLAVINE HYDROCHLORIDECETRIMONIUM BROMIDE CHELIDONINE (+) COLCHICEINE COLCHICINE CYTARABINEDACTINOMYCIN DEOXYSAPPANONE B 7.3′-DIMETHYL ETHER DIGITOXINDIHYDROGAMBOGIC ACID DISULFIRAM EMETINE GENTIAN VIOLET JUGLONELANATOSIDE C LYCORINE MITOMYCIN C OXYPHENBUTAZONE PATULIN PERIPLOCYMARINPERUVOSIDE PHENYLMERCURIC ACETATE PUROMYCIN HYDROCHLORIDE PYRITHIONEZINC PYRROMYCIN RETUSOQUINONE SANGUINARINE SULFATE SARMENTOGENINSTROPHANTHIDIN THIMEROSAL TOMATINE

TABLE 11 33 hits that specifically target NB12: 10-HYDROXYCAMTOTHECIN4′-DEMETHYLEPIPODOPHYLLOTOXIN ANDROGRAPHOLIDE AMODIAQUINEDIHYDROCHLORIDE AMSACRINE HYDROCHLORIDE ANCITABINE HYDROCHLORIDEBENZALKONIUM CHLORIDE BENZETHONIUM CHLORIDE BEPRIDIL HYDROCHLORIDEbeta-PELTATIN CAMPTOTHECIN CETYLPYRIDINIUM CHLORIDECHOLESTAN-3beta.5alpha.6beta-TRIOL CICLOPIROX OLAMINE CONVALLATOXINCRASSIN ACETATE CRINAMINE DIGOXIN ERYSOLIN GAMBOGIC ACID IMIDACLOPRIDELIMONIN MECHLORETHAMINE MECLIZINE HYDROCHLORIDE OUABAIN OXYBENDAZOLEPACLITAXEL PARAROSANILINE PAMOATE PARTHENOLIDE PODOPHYLLOTOXIN ACETATESTROPHANTHIDINIC ACID LACTONE ACETATE TENIPOSIDE VINBLASTINE SULFATE

TABLE 12 32 Compounds selected for IC50 Determinations: Aklavinehydrochloride AMSACRINE HYDROCHLORIDE ANCITABINE HYDROCHLORIDEANDROGRAPHOLIDE BEPRIDIL HYDROCHLORIDE beta-PELTATIN CGP-74514Ahydrochloride CHOLESTAN-3beta.5alpha.6beta-TRIOL CICLOPIROX OLAMINECONVALLATOXIN CRASSIN ACETATE CRINAMINE DIHYDROGAMBOGIC ACID ERYSOLINGambogic Acid IMIDACLOPRIDE JUGLONE LIMONIN MECHLORETHAMINE MECLIZINEHYDROCHLORIDE Mitomycin C Mitoxantrone hydrochloride OUABAINOXYBENDAZOLE PARAROSANILINE PAMOATE PERIPLOCYMARIN PERUVOSIDE PrenyletinPYRITHIONE ZINC TENIPOSIDE Tomatidine hydrochloride TOMATINE

These results suggest that patient-specific therapeutics, as well as themolecular and biochemical alterations that lead to NB, can be identifiedusing this assay.

Example 14 Identified Compounds that Affect Normal, Neuroblastoma orNeuroblastoma and Normal (Non-Neuroblastoma) Cells

The present example provides a description of the screening method usedto identify and select chemical entities capable of affecting (i.e.,reducing and/or inhibiting) NB cells. The screening method is used herewith the LOPAC™ compound collection. (LOPAC™ library, Sigma).

Candidate NB TICs were isolated. These TICs were used in the screeningassay for the identification of these kinds of compounds because theyare related to SKPs. For example, both SKPs and TICs originate from theneural crest, express similar neural crest markers, and differentiate invitro into similar cell types. The availability of two neural crest stemcell sources, one from the NB tumor and the other from the skin of thesame patient, affords an approach for the therapeutic target discoveryprovided here.

Materials and Methods: Methods:

To identify compounds that specifically target NB TICs, a cell-basedassay in which TICs from a NB patient and normal human pediatric SKPswere tested in parallel. Cells were treated with test compound prior toincubation with a cell viability dye. For both cell sources, signalvariability was low and the Z′ and Z factors were >0.5, suggestingexcellent assay quality. Hits were defined as compounds whose signalswere shifted at least 3 standard deviations from the mean.

Results:

Compounds that affect Neuroblastoma Cells and Normal Cells

From 3 libraries of compounds, the LOPAC™ collection, the PRESTWICKCHEMICAL LIBRARY® Collection and the SPECTRUM™ Collection, 46 compoundswere found to affect both normal and NB cells. These 46 compounds arelisted in Table 13.

TABLE 13 Normal and Neuroblastoma Hits 3-METHYLORSELLINIC ACID5alpha-CHOLESTAN-3beta-OL-6-ONE 5-AZACYTIDINE AKLAVINE HYDROCHLORIDEAlexidine dihydrochloride Ammonium pyrrolidinedithiocarbamate AnisomycinBrefeldin A from Penicillium brefeldianum Calmidazolium chlorideCephaeline dihydrochloride heptahydrate CETRIMONIUM BROMIDE CHELIDONINE(+) COLCHICEINE DACTINOMYCIN Daunorubicin hydrochloride DEOXYSAPPANONE B7.3′-DIMETHYL ETHER Digitoxigenin Digoxin DIHYDROGAMBOGIC ACIDDihydroouabain Disulfiram EMETINE GENTIAN VIOLET JUGLONE LANATOSIDE CLYCORINE Methyl benzethonium chloride MITOMYCIN C MitoxantroneOXYPHENBUTAZONE PATULIN PERIPLOCYMARIN PERUVOSIDE PHENYLMERCURIC ACETATEProscillaridin A Puromycin dihydrochloride PYRITHIONE ZINC PYRROMYCINRETUSOQUINONE Sanguinarine SARMENTOGENIN Strophanthidin TerfenadineTHIMEROSAL Thonzonium bromide TOMATINETable 14: 54 Identified Compounds that Affect NB Cells

Fifty-four (54) compounds selected from the LOPAC™ collection, PRESTWICKCHEMICAL LIBRARY® Collection and the SPECTRUM™ Collection, were found toselectively target NB cells. These 54 compounds appear in Table 14.

TABLE 14 Neuroblastoma Specific Hits 10-HYDROXYCAMTOTHECIN2.3-Dimethoxy-1.4-naphthoquinone 4′-DEMETHYLEPIPODOPHYLLOTOXINAmodiaquin dihydrochloride dihydrate AMSACRINE HYDROCHLORIDE Ancitabinehydrochloride ANDROGRAPHOLIDE Antimycin A Azaguanine-8 BENZALKONIUMCHLORIDE Benzethonium chloride BEPRIDIL HYDROCHLORIDE beta-PELTATINCamptothecin (S.+) CETYLPYRIDINIUM CHLORIDE CGP-74514A hydrochlorideChelerythrine chloride CHOLESTAN-3beta.5alpha.6beta-TRIOL Ciclopiroxethanolamine Clofazimine CONVALLATOXIN CRASSIN ACETATE CRINAMINECycloheximide Cytosine-1-beta-D-arabinofuranoside hydrochlorideDequalinium analog. C-14 linker Dequalinium dichlorideDiphenyleneiodonium chloride DL-Stearoylcarnitine chloride Doxorubicinhydrochloride ERYSOLIN Etoposide GAMBOGIC ACID Idarubicin IMIDACLOPRIDELIMONIN Loratadine MECHLORETHAMINE MECLIZINE HYDROCHLORIDE MG 624Mycophenolic acid Ouabain OXYBENDAZOLE Paclitaxel PARAROSANILINE PAMOATEParthenolide Podophyllotoxin Primaquine diphosphate Quinacrinedihydrochloride Scoulerine Taxol TENIPOSIDE Vinblastine sulfate saltVincristine sulfate

Four (4) compounds selected from the LOPAC™ collection, PRESTWICKCHEMICAL LIBRARY® Collection and the SPECTRUM™ Collection, were found tosuccessfully treat a NB patient and were selected as NB specific hitsaccording to the assay criteria provided herein. These compounds serveas positive controls in the selection and screening methods. Theseresults emphasize the validity of the assay in identifying active agentsfor treating NB. These 4 compounds are listed in Table 15.

TABLE 15 4 Identified Compounds that are used to Treat the NeuroblastomaPatient Patient Hits (i.e. drugs that were used to treat patient ANDwere selected as NB specific hits) Ancitabine hydrochloride (akacyclocytidine) Doxorubicin hydrochloride (aka adriamycin) EtoposideVincristine sulfate

These results suggest that patient-specific therapeutics as well asnovel molecular effectors of NB can be identified using this assay.

Example 15 Cumulative Screening Assay Selection Results

The present example presents the tabulated data obtained with thevarious chemical library screens conducted.

TABLE 16 NB NB + hit FS IC50 Library Name Repeated only hit test?target/mechanism S 10-HYDROXYCAMTOTHECIN X X modified camptothecin L2.3-Dimethoxy-1.4-naphthoquinone X X ROS modulator/Redox cycling agentused to study role of ROS S 3-METHYLORSELLINIC ACID X X Aspergillusterreus fungal metabolite; possible antioxidant S 4′- X XDEMETHYLEPIPODOPHYLLOTOXIN S 5alpha-CHOLESTAN-3beta-OL-6- X XCholesterol oxidation ONE product; cytotoxic due to oxidative stress orcytoskeleton disruption S 5-AZACYTIDINE X X S ACRIFLAVINIUMintercalating agent that HYDROCHLORIDE interferes with DNA replic/transcription; antitumor, antiproliferative S ACRISORCIN topicalanti-infective from 1960s S AKLAVINE HYDROCHLORIDE X X X naturalproduct; anti- infective; related structures have broad activity againstNIH tumor lines P Alexidine dihydrochloride X X phospholipase inh; oralgingivitis rinse S ALEXIDINE HYDROCHLORIDE P Alprostadil vasodilator;erectile dysfunction, palliative care for neonatal congenital heartdefects L Ammonium X X X blocks NOS mRNA pyrrolidinedithiocarbamatetranslation P !Amodiaquin dihydrochloride X X X antimalarial; treatmentof dehydrate CNS degeneration (Alzheimer, MS) S !AMODIAQUINE X Xantimalarial; 4- DIHYDROCHLORIDE aminoquinoline family; narrowtherapeutic/toxic window in children S AMSACRINE HYDROCHLORIDE X X Xtopo II inh; used in AML; may also be active vs malaria L Ancitabinehydrochloride X X cyclocytidine HCl; DNA- synthesis inhibitor (cytosineanalog); antileukemic S ANCITABINE X X X HYDROCHLORIDE S ANDROGRAPHOLIDEX X X Chinese herbal medicine; anti-inflamm; immune boosting?;anti-cancer vs HL60, MCF7, others through G0/G1 block and apoptosisinduction P Anisomycin X X protein synthesis inh thu peptidyltransferase of 80S ribosome; treatment activates p54, MAPK, SAPK P#Antimycin A X X X antifungal, antimicrobial; blocks e-transport betweencytochrome B and cytochrome C; bind the BH3 domain of Bcl-xL and induceapoptosis in cells overexpressing Bcl-2 and Bcl-xL P !Avermectin B1 Xantiworm/insecticide P Azaguanine-8 X purine analog S BENZALKONIUMCHLORIDE X X cationic detergent; v common antiseptic and preservative P+Benzethonium chloride X X topical antimicrobial used in cosmetics aspreservative S +BENZALKONIUM CHLORIDE X X S !BEPRIDIL HYDROCHLORIDE X XX nonselective Ca channel blocker used for treatment of chronic anginapectoris; alters potential dep and receptor-operated Ca channels andinhibits fast Na inward currents S beta-PELTATIN X X X extracted fromMayapple rhizome (like podophyllotoxin); some evidence of in vitroantitumor f/x but vague L Brefeldin A from Penicillium X X fungalmetabolite that brefeldianum disrupts Golgi structure and function LCalmidazolium chloride X X Potent inhibitor of calmodulin activation ofphosphodiesterase; strongly inhibits calmodulin- dependent Ca2+-ATPase SCAMPTOTHECIN X X L Camptothecin (S.+) X X topo 1 inh P Camptothecine(S.+) X X X P Cephaeline dihydrochloride X X ipecac alkaloidheptahydrate S CETRIMONIUM BROMIDE X X cationic detergent; quaternaryammonium compound used in hair conditioner and as a antimicrobial;tested as a lavage during colon resections . . . no benefit andpotentially toxic S CETYLPYRIDINIUM CHLORIDE X X active ingredient inScope; antiseptic used in oral rinses L CGP-74514A hydrochloride X X XCdk1 inh L Chelerythrine chloride X X X PKC inhibitor; affectstranslocation of PKC from cytosol to plasma membrane S CHELIDONINE (+) XX G2/M arrest associated with increased cycB1 levels, cdc2 activity andSAPK/JNK activity; weak tubulin interaction; induced apoptosis at 1uM inJurkat cells S CHOLESTAN-3beta.5alpha.6beta- X X X Cholesterol oxidationTRIOL product; cytotoxic due to oxidative stress or cytoskeletondisruption P Ciclopirox ethanolamine X X topical antifungal,antiinflammatory S CICLOPIROX OLAMINE X X X P Clofazimine X X X leprosytreatment; anti- inflammatory f/x; disrupts cc by binding DNA, may bindK+ transporters S COLCHICEINE X X metabolite of colchicine; less toxicto hepatocytes; less binding to tubulin but presumably has similar modeof action L Colchicine X X binds tubulin/blocks mitosis by preventingspindle formation; bioactive doses would be toxic P Colchicine X X X SCOLCHICINE S #CONVALLATOXIN X X X derived from lily of the valley;digitalis-like action S CRASSIN ACETATE X X X antineoplastic vs P388leukemia and HT29 colon cancer cells in vitro; extracted from marineinvertebrates S CRINAMINE X X X P Cycloheximide X X protein synthesisinh S CYCLOHEXIMIDE S CYMARIN X S CYTARABINE X Ara-C; DNA damage, S-phase block; inh DNA/RNA pol L Cytosine-1-beta-D- X X Ara-C; selectiveinh of arabinofuranoside hydrochloride DNA synthesis S DACTINOMYCIN X XP Daunorubicin hydrochloride X X DNA intercalator; neuroblastomatreatment S DEOXYSAPPANONE B 7.3′- X X flavanoid derived from DIMETHYLETHER Caesalpinia sappan tree; Chinese med treatment for tumor,diarrhea; aldose reductase inhibitor?; one study suggesting activity vshead and neck cancer cell line L Dequalinium analog. C-14 linker X X XProtein kinase C-alpha (PKC-alpha) inhibitor P !Dequalinium dichloride XX X Selective blocker of apamin-sensitive K+ channels L !Dequaliniumdichloride X X X Member of delocalized lipophilic cations (DLCs), afamily of compounds that accumulate in mitochondria driven by thenegative transmembrane potential; inhibitor of NADH- ubiquinonereductase; A novel mitochondria delivery system is based on dequalinium.This DLC forms liposome-like aggregates termed ‘DQAsomes’. DQAsomes arebeing tested as mitochondria drug delivery systems for small moleculessuch as paclitaxel P #Digitoxigenin X X Digitalis derivative; blocksNa+/K+ pump S #DIGITOXIN P #Digoxigenin X X Digitalis derivative; blocksNa+/K+ pump P #Digoxin X X X Digitalis derivative; blocks Na+/K+ pump S#DIGOXIN X X S DIHYDROGAMBOGIC ACID X X X L !Dihydroouabain X X X Na+/K+pump inhibitor L Diphenyleneiodonium chloride X X X eNOS inh(endothelial NOS) S DISULFIRAM X X P Disulfiram X X antabuse, rxn withalcohol use L DL-Stearoylcarnitine chloride X X X PKC inh P Doxorubicinhydrochloride X X DNA synthesis inh; stabilizes topo II complex afterstrand cleavage S EMETINE X X P Emetine dihydrochloride X X ipecacalkaloid; inh protein synthesis by blocking Rb movement on mRNA; inhibitDNA replication in S phase L Emetine dihydrochloride hydrate X XApoptosis inducer; RNA- Protein translation inhibitor S ERYSOLIN X X Xorganic isothiocyanate found in cruciferous veggies; increasesaccumulation of chemo drugs in PANC-1, MCF-7, NCI-H460 cell lines PEtoposide X X X topo II inh L Etoposide X X P Fosfosal salicylic acidderivative/ anti-inflammatory S GAMBOGIC ACID X X X principle pigment ofgambage resin (bright orange); caspase activator (not wellcharacterized); growth/tumor inhibitory vs HeLa, HEL, gastic cancer,lung carcinoma cell lines S GENTIAN VIOLET X X L Idarubicin X X Xantineoplastic, DNA metabolism S !IMIDACLOPRIDE X X X a4b2 nAChRagonist; activates ERK pathway; insecticide S JUGLONE X X X Pin1 inh;alkylates thioredoxin reductase; PI3K inh?; inhibits growth of HCT-15,HeLa, HL60 cell lines P Kaempferol antioxidant/flavenoid P #Lanatoside CX X Digitalis derivative; blocks Na+/K+ pump S #LANATOSIDE C X X SLIMONIN X X X isolated from citrus fruit seeds; inhibits HIV1 proteaseactivity; antinociceptive, inhibits MCF7 growth but not other cancercell lines L *Loratadine X X X H1 Histamine R antagonist S LYCORINE X XP +Mebendazole anthelmintic; blocks glucose/nutrient uptake in adultworm intestine; reported to be a mitotic spindle poison (resulting inchromosomal nondisjunction) S MECHLORETHAMINE X X X mustard gasderivative; polyfunctional alkylating agent = DNA breaks and crosslinks;non cc phase specific S *MECLIZINE HYDROCHLORIDE X X X antivert/bonine;motion sickness/vertigo treatment; piperazine class of antihistamines LMelphalan Antineoplastic; forms DNA intrastrand crosslinks bybifunctional alkylation in 5′-GGC sequences; used in NB megatherapy PMenadione vitamin K3 (vitK2 precursor); reacts with-SH/ soaks up GSH =high ROS = altered Ca2+ = Ca-dep DNA fragmentation; toxic at high dosesso vitK2 currently being used in cancer trials P +Methiazoleanthelmintic P +Methyl benzethonium X X topical antimicrobial S+METHYLBENZETHONIUM X X CHLORIDE L !MG 624 X X X Nicotinic acetylcholinereceptor antagonist; selectively inhibits alpha- bungarotoxin sensitivereceptors that contain the alpha7 subunit S MITOMYCIN C X X X SMITOXANTHRONE X HYDROCHLORIDE L Mitoxantrone X X topo II inh; used inALL, breast cancer, non- hodgkin's lymphoma P Mitoxantronedihydrochloride X X P Mycophenolic acid X X X immunosuppressant; blocksde novo purine biosynthesis S NERIIFOLIN L #Ouabain X X Blocks movementof the H5 and H6 transmembrane domains of Na+-K+ ATPases S #OUABAIN X XX S +OXYBENDAZOLE X X X benzimidazole anthelmintic used in horses andother ruminants S OXYPHENBUTAZONE X X Anti-inflammatory (Tandearil);binds phospholipase A2, human neutrophil elastase P Paclitaxel X X Xtaxol S PACLITAXEL X X taxol S PARAROSANILINE PAMOATE X X X PParthenolide X X X feverfew extract; NFkB inh, p53 activ, increased ROS,JNK activ (indep of NFkB and ROS), inh of MAPK/ERK pathway SPARTHENOLIDE X X seems to work best as a chemosensitizer . . . studiesin breast, skin, pancreatic, thoracic cell lines S PATULIN X X S#PERIPLOCYMARIN X X X digoxin relative S #PERUVOSIDE X X X inhibitor ofNa+K+- ATPase; cardiac glycoside class S PHENYLMERCURIC ACETATE X X PPodophyllotoxin X X X etoposide precursor/ Antineoplastic glucoside;inhibitor of microtubule assembly; G2/M cc arrest L Podophyllotoxin X XS PODOPHYLLOTOXIN X X ACETATE P !Primaquine diphosphate X X Xantimalarial/inh of DNA, RNA, protein synthesis/ muscarinic AChR inh P#Proscillaridin A X X Na+/K+ ATPase inh; digitalis related P Puromycindihydrochloride X X protein synthesis inh, premature strand terminationS PUROMYCIN X X HYDROCHLORIDE S PYRITHIONE ZINC X X X S PYRROMYCIN X Xanthracycline derivative; monosaccharide; induces erythroid diff in K562P +Pyrvinium pamoate pinworm treatment; prevents gluccose uptake;antitumor activity vs pancreatic cell line in SCID model, see decreaseAkt phos L !Quinacrine dihydrochloride X X X Monoamine oxidase (MAO)inhibitor; antimalarial P !Quinacrine dihydrochloride X X Antimalarial,causes female dihydrate sterility S RETUSOQUINONE X X ? P !SanguinarineX X Inhibitor of Mg2+ and Na+/K+-ATPase; isolated from the leaves andstems of Macleaya cordata and microcarpa L !Sanguinarine chloride X X XS !SANGUINARINE SULFATE X X S SARMENTOGENIN X X P !Scoulerine X X Xopium intermediate/alkaloid; a1- adrenoreceptor inh (G- protein coupledR found on PNS sympathetic nerve terminals, CNS postsynaptically; targetof catecholamines) P !Strophanthidin X X X blocks Na+/K+ ATPase at highconc; opposite f/x at low dose (Quabain) S !STROPHANTHIDIN X X S!STROPHANTHIDINIC ACID X X !LACTONE ACETATE P !Strophantine octahydrateX X L Taxol X X Antitumor agent; promotes assembly of microtubules andinhibits tubulin disassembly process S TENIPOSIDE X X X common NBtreatment; semisynthetic podophyllotoxin derivative related toetoposide; topo II inh; induced single strand DNA breaks; activity inlate S and G2 phases P *Terfenadine X X X nonsedating antihistimineoffmarket due to cardiac f/x S THIMEROSAL X X S THIRAM P Thonzonium bromideX X cationic detergent S TOMATINE X X X P Verteporfin photoreactive dyeused in treatment of macular generation; anti-angiogenic S VINBLASTINESULFATE X X L Vinblastine sulfate salt X X X Inhibitor of microtubuleassembly L Vincristine sulfate X X X Inhibitor of microtubule assembly132/151 repeated (87%) BOLD: DNA damage/protein synthesis inhibitor/cellcycle block italics: protein inhibitor/activator *antihistamine #digoxinderivative +: metabolic f/x !ion channel inhibitor/neuro R inhibitor

Example 16 Selected Compounds of Interest

The present example demonstrates the utility of the present inventionfor providing a composition suitable for the inhibition of NB survival,proliferation, or induction of differentiation, and for the treatment ofNB.

Forty-seven (47) compounds were selected based on differential celltoxicity and compound mechanism of action. Forty are novel compounds forthe treatment of NB. None of these 40 compounds have been usedclinically in NB therapy nor have they been examined in clinical trials.Seven compounds have been previously used for NB treatment (marked withasterisk), and serve as positive controls in the selection and screeningprocess of new chemical entities that may be used in the treatment of NBaccording the present invention.

TABLE 17 NB12 IC50 Compounds of Interest: (nM) Notes:2.3-Dimethoxy-1.4-naphthoquinone nd ROS modulator/Redox cycling agentused to study role of ROS AKLAVINE HYDROCHLORIDE 778.5 natural product;anti-infective; related structures have broad activity against NIH tumorlines Amodiaquin dihydrochloride dehydrate 790 antimalarial; treatmentof CNS degeneration (Alzheimer, MS); 4-aminoquinoline family; narrowtherapeutic/toxic window in children; 4-Aminoquinolines depress cardiacmuscle, impair cardiac conductivity, and produce vasodilatation withresultant hypotension AMSACRINE HYDROCHLORIDE 1214 topo II inh; used inAML; may also be active vs malaria *ANCITABINE HYDROCHLORIDE 519.7cyclocytidine HCl; DNA-synthesis inhibitor (cytosine analog);antileukemic Azaguanine-8 331 purine analog beta-PELTATIN 1949 extractedfrom Mayapple rhizome (like podophyllotoxin); some evidence of in vitroanti-tumor f/x but vague Camptothecine (S.+) 183.3 topoisomerase 1 inhCGP-74514A hydrochloride Cdk1 inh Chelerythrine chloride 2553 PKCinhibitor; affects translocation of PKC from cytosol to plasma membraneCHOLESTAN-3beta.5alpha.6beta- 2410 Cholesterol oxidation product;cytotoxic due to oxidative TRIOL stress or cytoskeleton disruptionCICLOPIROX OLAMINE 2048 topical antifungal, anti-inflammatory viainhibition of 5- lipoxygenase and cyclooxygenase; hydroxypyridonefamily; Loprox Clofazimine 1417 leprosy treatment; anti-inflammatoryf/x; disrupts cc by binding DNA, may bind K+ transporters Colchicine29.3 binds tubulin/blocks mitosis by preventing spindle formation;bioactive doses would probably be toxic CONVALLATOXIN 73.17 derived fromlily of the valley; digitalis-like action CRASSIN ACETATE 1947antineoplastic vs P388 leukemia and HT29 colon cancer cells in vitro;cembranolides (14-member ring diterpenoid lactones) derived fromCaribbean gorgonians (marine invertebrates) CRINAMINE 1735 HIF-1alphainhibitor; affinity to the serotonin reuptake transport proteinDequalinium analog. C-14 linker 1112 Protein kinase C-alpha (PKC-alpha)inhibitor Dequalinium dichloride 3617 Selective blocker ofapamin-sensitive K+ channels; mitochondria toxicity Digitoxin nd Na+/K+pump inhibitor Digoxigenin nd Na+/K+ pump inhibitor Digoxin 542.2Digitalis derivative; blocks Na+/K+ pump DIHYDROGAMBOGIC ACID 1687Dihydroouabain 1540 Na+/K+ pump inhibitor ERYSOLIN 3276 organicisothiocyanate found in cruciferous veggies; increases accumulation ofchemo drugs in PANC-1, MCF- 7, NCI-H460 cell lines *Etoposide 693.7topoisomerase II inh GAMBOGIC ACID 1695 principle pigment of gambageresin (bright orange); caspase activator (not well characterized);growth/tumor inhibitory vs HeLa, HEL, gastic cancer, lung carcinoma celllines *Idarubicin 203.7 antineoplastic, DNA metabolism MECHLORETHAMINE438.2 mustard gas derivative; polyfunctional alkylating agent = DNAbreaks and crosslinks; non cell cycle phase specific MECLIZINEHYDROCHLORIDE 2537 “antivert/bonine”; motion sickness/vertigo treatment;piperazine class of antihistamines MG 624 848 Nicotinic acetylcholinereceptor antagonist; selectively inhibits alpha-bungarotoxin sensitivereceptors that contain the alpha7 subunit MITOXANTHRONE 60.46 topo IIinh; used in ALL, breast cancer, non-hodgkin's HYDROCHLORIDE lymphomaOUABAIN 122.6 Blocks movement of the H5 and H6 transmembrane domains ofNa+-K+ ATPases OXYBENDAZOLE nd benzimidazole anthelmintic used in horsesand other ruminants Paclitaxel nd aka taxol; Antitumor agent; promotesassembly of microtubules and inhibits tubulin disassembly processParthenolide 2261 feverfew extract; NFkB inh, p53 activ, increased ROS,JNK activ (indep of NFkB and ROS), inh of MAPK/ERK pathway; seems towork best as a chemosensitizer...studies in breast, skin, pancreatic,thoracic cell lines PATULIN nd polyketide lactone, produced by certainfungal species of Penicillium, Aspergillus and Byssochlamys growing onfruit, including apples, pears, grapes; crosslinks DNA, causes p38 andJNK phosphorylation in HEK cells PERIPLOCYMARIN 2703 digoxin relativePERUVOSIDE 222.5 inhibitor of Na+K+-ATPase; cardiac glycoside class*Podophyllotoxin 135 etoposide precursor/Antineoplastic glucoside;inhibitor of microtubule assembly; G2/M cc arrest Primaquine diphosphatend antimalarial/inh of DNA, RNA, protein synthesis/ muscarinic AChR inhQuinacrine dihydrochloride 2556 Monoamine oxidase (MAO) inhibitor;antimalarial Sanguinarine chloride 1795 Inhibitor of Mg2+ andNa+/K+-ATPase; isolated from the leaves and stems of Macleaya cordataand microcarpa *TENIPOSIDE 705.5 common NB treatment; semisyntheticpodophyllotoxin derivative related to etoposide; topo II inh; inducedsingle strand DNA breaks; activity in late S and G2 phases TOMATINE ndalkaloid found in leaves of tomato and unripe fruit; tetrasaccharidetomato glycoalkaloid alpha-tomatine, trisaccharide beta(1)-tomatine,disaccharide gamma- tomatine, monosaccharide delta-tomatine, and theircommon aglycon tomatidine; inhibit the growth of human colon (HT29) andliver (HepG2) cancer cells *Vinblastine sulfate salt 113 Inhibitor ofmicrotubule assembly *Vincristine sulfate 61.95 Inhibitor of microtubuleassembly

Tables 7, 11, 14, and 17 identify parthenolide, a compound thatspecifically targets NB TICs, as a compound previously identified totarget human acute myelogenous leukemia stem and progenitor cells (55).

Example 17 Secondary Screening

In vitro testing of compounds of interest will be conducted on NB TICsfrom different patients using a methylcellulose assay (or 96 well liquidculture) with drug dose response curve to assess stem cell killing,proliferation, or differentiation, and conducted on the following celllines:

NB12, NB10 (GN), NB19, NB25, NB05 (TICs or primary sphere-forming cellsfrom NB patients)

FS90, FS105, FS99, FS107, FS81 (pediatric human SKPs)

KCNR (human established NB cell line)

In addition, a combination treatment with the most promising compoundsand currently-used chemotherapeutic agents will be conducted determineif compounds of interest will act synergistically with thecurrently-used compounds to induce cell death, stop cell proliferation,or induce differentiation into neural cell types.

Chemical Optimization

Structurally similar compounds to those identified above will be testedto determine if they will induce TIC or NB death at lower effectivedoses.

Animal Models

TICs will be injected orthotopically into the mouse adrenal fat pad (thesite of the majority of human NB), tumors allowed to initiate for sevendays, mice injected with a range of doses of compounds, and suppressionof tumor size determined by histological analysis andimmunohistochemistry for TH and NB84, for tumor cell death by theexpression of cell death markers cleaved caspase and by TUNEL assay,cessation of cell proliferation by anti-MIB-1 immunohistochemistry, andinhibition of metastasis by histological and immunohistochemicalexamination of liver, bone marrow, and kidney by anti-NB84 and tyrosinehydroxylase.

TICs will be injected into the mouse inguineal fat pad, tumors allowedto initiate for seven days, and mice injected with a range of doses ofcompounds, tumors allowed to initiate for seven days, mice injected witha range of doses of compounds, and suppression of tumor size determinedby histological analysis and immunohistochemistry for TH and NB84, fortumor cell death by the expression of cell death markers cleaved caspaseand by TUNEL assay, cessation of cell proliferation by anti-MIB-1immunohistochemistry, and inhibition of metastasis by histological andimmunohistochemical examination of liver, bone marrow, and kidney byanti-NB 84 and TH.

TICs will be injected into mice that have NB as a result of expressionof the MYCN oncogene (31), tumors allowed to initiate for seven days,and mice injected with a range of doses of compounds, tumors allowed toinitiate for seven days, mice injected with a range of doses ofcompounds, and suppression of tumor size determined by histologicalanalysis and immunohistochemistry for TH and NB84, for tumor cell deathby the expression of cell death markers cleaved caspase and by TUNELassay, cessation of cell proliferation by anti-MIB-1immunohistochemistry, and inhibition of metastasis by histological andimmunohistochemical examination of liver, bone marrow, and kidney byanti-NB84 and TH.

Example 18 Tumor Initiating Cells in Childhood Neuroblastoma

The present example demonstrates the utility of the present inventionfor providing a method for providing an enriched population of human NBTICs, and in particular, from a child having NB. In addition, thepresent example demonstrates the utility of The present exampledemonstrates the utility of the present invention for providing presentinvention for providing a method by which therapeutic agents suitablefor the treatment of a human, particularly a child, having NB may beselected.

Materials and Methods:

Primary Culture of Tumor Spheres from Tumors and Bone Marrow Aspirates

Tumor samples and bone marrow aspirates were obtained from consentedpatients, as approved by the Hospital for Sick Children's ResearchEthics Board. Bone marrow aspirates were filtered through a 40-μm cellstrainer and tumor cells collected by inverting and washing the filterwith Hank's balanced salt solution (HBSS; Invitrogen, Carlsbad, Calif.),while tumor samples were collected and cut into 2-3 mm² pieces. Allsamples were then enzymatically dissociated with LIBERASE® Blendzyme 1(an enzyme blend) (0.62 Wunsch U/ml; Roche, Indianapolis, Ind.) in HBSSfor 15-45 minutes at 37° C. and 10% Fetal Bovine Serum (FBS; HyClone,Logan, Utah) added to inhibit enzyme activity. Tumor cells were thenmechanically dissociated in medium and the suspension poured through a40 μm cell strainer. Dissociated cells were pelleted and resuspended inDulbecco's modified Eagle's medium [DMEM-F12 (3:1) (Invitrogen)containing 1% penicillin/streptomycin, 2% B27 supplement (Gibco,Carlsbad, Calif.), 40 ng/ml FGF and 20 ng/ml EGF (both fromCollaborative Research, Bedford, Mass.), from herein referred to asproliferation media, and cultured in 25 cm² flasks (Falcon) in a 37° C.,5% CO₂ tissue-culture incubator. Cells were fed fresh proliferationmedium weekly.

Tumor spheres were passaged by mechanical dissociation and split 1:6with 50% fresh proliferation medium and 50% conditioned medium from theinitial flask. Human SKPs, which are of neural crest origin, were usedas normal comparative cells in our experiments, and were isolated andcultured according to protocols established in the laboratory (18).

Tumor Sphere Self-Renewal Assay and Growth Curves

Self-renewal is a fundamental feature of stem cells, either of normal ortumor origin, and can be tested by serial passage (58, 14). Theself-renewal capacity of tumor spheres was assesed in the presentexample using a semi-solid methylcellulose medium. After primary spheresformed, spheres were mechanically dissociated into single cells andplated into uncoated 24-well tissue culture plates (Falcon) containing 1ml of 0.9% methylcellulose (Sigma, St Louis, Mo.), 10% conditionedmedium and growth factors as described for the proliferation medium.Final plating densities ranged from 25 000 cells/ml to 100 cells/ml.Cultures were fed 150 μl proliferation media twice weekly for 21 days,when the number of spheres for each plating density was counted.Self-renewal capacity was calculated as the percentage of single cellsthat were able to form spheres.

Growth curves were established by mechanically dissociating passagedtumor spheres, plating 8.3×10⁴ single cells in 12.5 cm² flasks andperforming cells counts 3, 5 and 7 days after plating. The mean cellcount of 3 independent experiments was graphed and SEM calculated.

Neurogenic Differentiation of Tumor Spheres

Five to ten tumor spheres were plated onto poly-D-lysine/laminin-coated8-well chamber slides (Nalge Nunc, Rochester, N.Y.) expanded in mediumcontaining DMEM-F12 (3:1), 10 ng/ml FGF and 15% FBS for 5-10 days andthen differentiated in Neurobasal medium (Invitrogen) containing 2% B27supplement, 1% FBS, 1% N2 supplement (Gibco), 16 μg/ml NGF (Cedarlane,Hornby, ON), and 8 ng/μl NT3 (Peprotech, Rocky Hill, N.J.) for a further14 days. Half media was replaced every second day throughout the assay.

Immunocytochemistry and Quantification

Immunocytochemical analysis of cells was performed using coated glassslides and the Shandon CYTOSPIN® system (a cytocentrifuuge) (Thermo,Waltham, Mass.) for tumor spheres or cells differentiated on chamberslides as described (16; 18). The following primary antibodies wereused: NB84 monoclonal (1:50; Novocastra, Newcastle upon Tyne, UK);anti-THpolyclonal (1:150; Chemicon, Temecula, Calif.); anti-βIII-tubulinmonoclonal (1:500; Tuj1 clone; Covance, Berkeley, Calif.);anti-neurofilament-M polyclonal (NFM) (1:200; Chemicon); s10013monoclonal (1:1000; Sigma); anti-GFAP polyclonal (1:200; DAKO,Copenhagen, Denmark); Galactocerebroside C (GalC) polyclonal (1:200;Chemicon); anti-Nestin monoclonal (1:400; Chemicon); anti-Nestinpolyclonal (1:400, Chemicon); anti-fibronectin polyclonal (1:400;Sigma). The following secondary antibodies were used: Alexa488-conjugated goat-anti-mouse (1:1000) and Alexa 555-conjugatedgoat-anti-rabbit (1:1000), both were from Molecular Probes (Eugene,Oreg.).

Differentiation was quantified by calculating the percentage of spheresthat formed either neuronal networks or neurons. Data was pooled forgood (stages 1-3 and 4S) and poor prognosis (stage 4) NB tumors and theoverall mean and SEM calculated.

Orthotopic Assay of In Vivo Tumorigenicity and Immunohistochemistry

Four to 5 week-old female SCID/Beige mice (Taconic; Hudson, N.Y.) werehoused in pathogen-free conditions and cared for in accordance with theNational Institutes of Health Animal Care and Use Committee. Animalswere acclimatized for 1 week prior to surgery. Surgical sites wereprepared by shaving and cleansing with Betadine scrub solution and 70%sterile alcohol. Anesthesia was induced using 5% isoflurane/1.5 L oxygenand maintained 3% isoflurane/1.5 L oxygen inhalation. In vitro passagedprimary NB cells (passages 4-5) were harvested and brought to final celldensities of 3×10⁶, 3×10⁵ or 3×10³/ml in HBSS for both orthotopicadrenal and heterotopic subcutaneous injections. Cells were kept at 4°C. until ready for injection and mixed 1:3 with basement membraneextract (Trevigen, Gaithersburg, Md.) just prior to injection (finalcell doses 10⁵, 10⁴ and 10²). Orthotopic and heterotopic injections wereperformed as previously described (28). Animals were monitored thriceweekly for evidence of tumor formation and associated morbidity.

All mice that were sacrificed underwent complete necropsy examinationand tissues fixed in 10% formalin for 24 hours prior to paraffinembedding and staining with hematoxylin and eosin (H&E) or a small pieceof tumor collected and re-implanted to follow secondary tumor formation.The endpoints evaluated were the percent tumor-take that is thepercentage of animals that developed primary tumors and tumor latency,which is the time from tumor cell injection to the detection of aprimary tumor.

The following antibodies were used for immunohistochemical analysis oforthotopic

adrenal tumors: NB84 monoclonal (1:20), anti-TH polyclonal (1:150), andanti-nestin:polyclonal (1:200). Sections were incubated with polyclonalswine anti-goat, mouse, rabbit biotinylated secondary antibody (1:500)(DAKO) and then tertiary staining was performed withperoxidase-conjugated strepavadin (1:500) (DAKO). Staining wasvisualized using 3,3′-diaminobenzidine (DAB) as a chromogen andcounterstained with hematoxylin.

Flow Cytometry and Fluorescence Activated Cell Sorting

Cultured primary NB cells were collected, washed twice in HBSS andresuspended as single cells in buffer (0.5% bovine serum albumin inPBS). If unconjugated primary antibodies were used, cells were initiallyblocked in human IgG (Jackson ImmunoResearch, West Grove, Pa.) and thenincubated in primary antibody for 30 minutes at 4° C. If an uncongugatedprimary antibody was used, cells were pelleted, resuspended in bufferand incubated with Alexa Fluor 488-conjugated goat anti-mouse secondaryantibody (Molecular Probes, Invitrogen) for 20 minutes at 4° C. Cellswere then washed twice in buffer and resuspended and fixed in buffer/2%paraformaldehyde. Approximately 10⁵ cells were stained and analyzed on aBecton Dickinson FACSCalibur 4-color analyzer.

Monoclonal antibodies against human CD133/1 (biotin) and CD271 (FITC)were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany); themonoclonal antibody against human CD56 (PE) was purchased from DAKO; themonoclonal antibody against humanNB84 (FITC) was purchased fromNovocastra; the monoclonal antibodies against human CD24 (PE), CD29(PECy5), CD81 (APC), CD34, CD44, CD45, CD20 and CD117, and ratmonoclonal antibody CD49f (PE) were purchased from BD biosciences(Oakville, ON). Isotype matched mouse or rat purified, PE- orFITC-conjugated antibodies (BD Biosciences) were used as controls.

Fluorescence-activated cell sorting (FACS) was done on a DAKOCYTOMATION® MOFLO® 9-color cell sorter. Sorting was performed on doublestained cells. Cells were stained with purified monoclonal CD34 andAlexa Fluor 488-conjugated goat anti-mouse secondary antibody followedby PE-conjugated monoclonal CD24 antibody.

Results:

Neuroblastoma Cells from Tumors and Bone Marrow Aspirates formNon-Adherent Spheres when Grown in Serum-Free Conditions:

Neuroblastoma cells from tumors and bone marrow aspirates formnon-adherent spheres when grown in serum-free conditions.

Seventeen tumor samples were used in the study including 6 high stage(stage 4) and 7 low stage NB tumors and 4 ganglioneuroma tumors, abenign cousin of NB (See Summary of Patient Population Table).

Summary of Patient Population Table PATIENT # SEX AGE AT DX. DX. SAMPLETYPE 1 F >18 m Stage 4 neuroblastoma Relapse bone marrow 2 F >18 mGanglioneuroblastoma Tumour 3 M >18 m Stage 4 neuroblastoma Relapse bonemarrow 4 F >18 m Ganglioneuroma Tumour 5 F >18 m Stage 4 neuroblastomaRelapse bone marrow 6 M >18 m Stage 1 neuroblastoma Tumor 7 F <18 mStage 1 neuroblastoma Tumor 8 F >18 m Ganglioneuroma Tumor 9 (matched to13) F >18 m Stage 1 neuroblastoma Bone marrow 10  M >18 m GanglioneuromaBone marrow 11  F >18 m Stage 2 neuroblastoma Tumor 12  M >18 m Stage 4neuroblastoma Tumor 13 (matched to 9)  F >18 m Stage 1 neuroblastomaTumor 14  M <18 m Stage 1/4S neuroblastoma Metastasis 15  M >18 m Stage4 neuroblastoma Relapse bone marrow 16  M >18 m Ganglioneuroma Tumor 17*F <18 m Stage 4 neuroblastoma Bone marrow *borderline MYCN-amplified,patient died.

A serum-free growth medium was used to isolate a putative TIC from theacutely dissociated tumor cells. Serum free growth medium favors stemcell growth (16, 18). Within 1-7 weeks (median time 2 weeks), a primarysphere formation was observed in the cultures as previously described(56). Samples that remained as single cells following acute dissociationand several weeks of culture were excluded from this study. Thesesamples included many low-grade (stage 1 and 4S) and some heavilytreated NB tumor samples.

Upon passaging, the majority of primary spheres from high-grade tumors(FIG. 5A) and bone marrow samples (FIG. 5B) formed secondary spheres.However primary ganglioneuroma tumor spheres (FIG. 5D) and primary tumorspheres from low-grade NB samples (FIG. 5E) tended to form adherentcultures when passaged. A single high-grade NB tumor sample becameadherent when primary tumor spheres were passaged (FIG. 5C).

To characterize and aid preliminary identification of the primary tumorspheres grown in serum-free media, clinically recognized standardneuroblastic markers NB84 and TH were used, and the characteristic SKPsprogenitor cell markers fibronectin and nestin were used as identifiersof neuroblastic progenitor origin prior to passaging and undertakingfurther characterization assays (FIG. 5F).

Low-Grade Tumor Neuroblastoma Spheres Exhibit Limited Potential forSelf-Renewal.

The self-renewal capacity of primary tumor spheres was evaluated bymechanical dissociation of the spheres and plating serial dilutions ofcells in semi-solid methylcellulose down to 100 cells/ml. All tumorsample that formed primary tumor spheres in liquid culture were testedin this manner. Tumor spheres from all NB phenotypes, ganglioneuromatumors and control SKPs formed secondary tumor spheres inmethylcellulose. Secondary tumor spheres and SKPs spheres weredissociated and re-plated in methylcellulose until sphereforming cellpopulations were depleted. Secondary spheres from low-grade NB andganglioneuroma tumors formed tumor spheres 0-6 more times (average3.91), high-grade NB samples formed tumor spheres 3-21 more times(average 8.00) when passaged in methylcellulose (FIG. 6A), and SKPsformed subsequent spheres 1-4 more times.

A minimum of three plating densities in the dilution series were countedto determine the average self-renewal for each passage. Linearregression analysis of plating densities showed that the number ofresultant spheres was proportional to the number of single cells plated,and did not alter with passaging (FIG. 6B). Growth curves were performedon high-grade tumor spheres showing high growth rates in culturedprimary tumors cells (patient 5, passage 7) (FIG. 6C).

The morphology of secondary and subsequent tumor spheres was identicalto that of primary spheres. Cells retained their expression of the NBmarkers NB84 and TH with passaging (FIG. 6D). All tumors studiedcontained a sub-population of cells that had the capacity to self-renew,however reflecting the clinical aggressiveness of the tumor phenotypes,aggressive NB tumors had increased potential to self-renew with threeprimary lines from high-grade NB tumor samples continuing to grow andexpand extensively.

High-Grade Tumor Spheres Exhibit Limited Differentiation Potential UnderNeurogenic Conditions

Neuroblastoma is a tumor resembling tissues derived from the embryonicneural crest; therefore conditions were used to differentiate SKPs, anormal neural crest-derived human precursor cell, to test themulti-potency of the primary tumor spheres isolated as described herein.(18). After differentiation in neurogenic conditions for two weeks,immunocytochemistry was performed on the differentiated tumor spheresusing dual sympathetic neuronal markers (TH, BIII-tubulin, NFM andnestin) or dual markers of glial cells (s100β, GFAP, GalC) to confirmidentity of arising cell types. Differentiated cells from both low andhigh-grade NB tumor spheres retained expression of the NB marker, NB84and the catecholamine biosynthetic pathway marker, a unique feature ofNB, TH with differentiation (FIG. 7A).

Ganglioneuroma tumor spheres and tumor spheres from all NB tumorphenotypes were capable of differentiating into neuronal lineagesexpressing nestin and βIII-tubulin, neuronal lineage marker commonlyobserved in peripheral neurons (FIG. 7B) (16; 17:18). The neurogenicconditions under which the tumor spheres were differentiated were notdesigned to promote differentiation of cells along glial lineages.However rare, spontaneous Schwannlike cells were observed thatimmunostained with the glial lineage markers s100β, GFAP and GalC, andshowed an appropriate phenotype in high-grade NB tumor spheres.Schwann-like cells were not observed in tumor spheres from low-grade NBor ganglioneuroma patients.

Differentiation assays were performed on whole tumor spheres underneuronal conditions and differentiation potential was determined bycalculating the percentage of tumor spheres that gave rise to TH,βIII-tubulin, NFM or nestin-positive neurons, either as individualneurons or large neuronal networks. High-grade NB tumor spheres (n=5)showed limited differentiation potential when compared to low-gradetumor spheres (n=5), specifically in their ability to form neuronalnetworks (FIG. 7C). Ganglioneuroma tumor spheres (n=4) showed similaroverall differentiation potential (mean±SEM) (34.97±19.85%) to low-gradeNB spheres (47.33±19.63%), but similarly low potential for formingneuronal networks (2.27±2.27%) as observed in high-grade NB tumorspheres (1.45±1.45%). Tumor spheres derived from the bone marrow of 1ganglioneuroma and 2 high-grade NB patients failed to differentiateunder any conditions (patients 5, 10 and 15).

Bone Marrow-Derived Tumor Spheres are a Naturally Enriched Source ofTumor-Initiating Cells.

In vivo assays have become the standard for evaluating both tumorpropagation and self-renewal (60). An orthotopic adrenal model forassessing tumor propagation was used with these cells in mice since NBtumors most frequently arise in the adrenal medulla.

Between 10² and 10⁵ dissociated high-grade NB tumor sphere cells wereinjected into the adrenal fat pads of immune-compromised mice and waiteduntil palpable tumors or tumor-associated morbidity was observed.Micro-tumors were observed in several animals injected with 10² cellswithin 3 weeks of injection by planned sacrifice and observed muchlarger tumors when a greater cell number was used (FIG. 8A).Furthermore, these tumors contained cells resembling immatureneuroblasts with small refractile cell bodies and a high nuclear tocytoplasmic ratio and stained positive for the clinical NB markers NB84,TH, and the stem cell marker nestin (arrow heads, FIG. 8A). High-gradeNB tumor sphere cells metastasized to distant sites including the lung,liver, spleen and contralateral adrenal and kidney and invaded localorgans (arrow heads, FIG. 8B), similar to tumor behavior in children.Time to morbidity was shorter with higher cell doses (FIG. 8C). Thepercentage of animals with evidence of tumors, and similarly those withdistant metastases increased according to cell dose (FIGS. 8D and 8E,respectively) the animals received. Tumors and metastases were notobserved when 10² cells were injected heterotopically intoimmune-compromised mice.

A small piece of in vivo tumor was taken at sacrifice and re-implantedinto immune-compromised mice to follow secondary tumor formation ofthese cells. Subsequent tumor formation was observed in two independenthigh-grade NB tumor spheres, suggesting long term self-renewal potentialof high-grade NB tumor spheres exists both in vitro and in vivo.

Tumor-Initiating Ability of High-Grade Tumor Spheres is Highly Enrichedin the CD24⁺/CD34⁺ Fraction of High-Grade Neuroblastoma Tumor Spheres.

Cells were negative for the previously published brain TIC markerCD133/1 (56) (FIG. 9A), and highly positive for the melanomatumor-initiating enrichment marker CD20 (61) the clinical NB marker NB84(FIG. 9A) and CD271/p75 (FIG. 9A), so were not used as candidate uniqueidentifiers in NB. The presence of a small fraction of CD24⁺ and CD34⁺cells was observed in our high-grade NB tumor spheres derived from bonemarrow aspirates (patients 5 and 14) (FIGS. 9B and 9D, respectively),that were otherwise absent in NB cell lines. and ganglioneuroma (patient4) tumor spheres. Further, bone marrow-derived high-grade NB tumorspheres were immunocytochemically stained for CD24 and CD34 confirmingthat a few cells within the tumor sphere that stained positive for ourpotential markers (FIGS. 9C and 9E, respectively).

The enrichment capacity of these markers was examined for tumorformation by orthotopic injection of each population of a CD24⁺/CD34⁺double sort (total, CD24⁻/CD34⁻, CD24⁺/CD34⁻, CD24⁻/CD34⁺, CD24⁺/CD34⁺cell populations). While all cellular fractions formed tumors inimmune-compromised mice, CD24⁺/CD34⁺ tumors formed in half the time ofall other cellular fractions (mean±SEM) (19.0±0.0 days compared with34.0±0.72 days) (FIG. 9F), suggesting that the CD24⁺/CD34⁺ enriches thetumor-forming potential of bone marrow-derived NB tumor spheres.

Example 19 Screening Kits and Personalized Medicine Cancer Stem CellScreening Kits

The present example is provided to demonstrate the utility of thepresent invention as providing a screening kit that may be used for theidentification of specific anti-NB TIC compounds and chemical entities.

The invention provides a kit for the testing and/or screening of apatient of interest's NB TICs. In this manner, a sample of biologicaltissue enriched for a population of NB TICs from a patient of interestmay be used to screen and/or identify a specific anti-NB TICs activeagent or agents that are the most potent and/or active against aspecific patient's NB TICs population. In this manner, potentialtherapeutic agents may be selected that is custom tailored to aparticular patient.

In some embodiments, the kit would comprise an assay plate that includesa plurality of wells, each well of said assay plate being suitable forcontaining a pharmacologically active agent of interest, such as apotentially anti-NB TICs pharmacologically active agent. By way ofexample, the assay plate may comprise 40, 50, 60, 70, 80, 90, 100 ormore wells. In some embodiments, the assay plate will include 96 wells,such as is customary in assay plates. As part of the kit describedherein, 5, 10, 20, 25, 30, or 40 of the wells may include a differentanti-NB TIC compound, such as a volume of one or more of each of thecompounds listed below:

-   -   2.3-Dimethoxy-1.4-naphthoquinone,    -   Aklavine Hydrochloride,    -   Amodiaquin dihydrochloride dehydrate;    -   Amsacrine Hydrochloride;    -   Azaguanine-8;    -   beta-peltatin;    -   Camptothecine (S.+);    -   CGP-74514A hydrochloride;    -   Chelerythrine chloride;    -   Cholestan-3beta.5alpha.6beta-Triol;    -   Ciclopirox Olamine;    -   Clofazimine;    -   Colchicine;    -   Convallatoxin;    -   Crassin Acetate;    -   Crinamine;    -   Dequalinium analog. C-14 linker;    -   Dequalinium dichloride;    -   Digitoxin;    -   Digoxigenin;    -   Dihydrogambogic acid;    -   Dihydroouabain;    -   Erysolin;    -   Gambogic acid;    -   Mechlorethamine;    -   Meclizine hydrochloride;    -   MG 624;    -   Mitoxanthrone Hydrochloride;    -   Ouabain;    -   Oxybendazole;    -   Oxybendazole;    -   Paclitaxel;    -   Parthenolide;    -   Patulin;    -   Periplocymarin;    -   Peruvoside;    -   Primaquine diphosphate;    -   Quinacrine dihydrochloride;    -   Sanguinarine chloride, or Tomatine.

In this manner, a positive control is provided in the assay plate forcontrol and/or comparative purposes.

In addition, and in some embodiments of the kit, at least one or more ofthe assay wells will include a volume of a pharmacologically activeagent that is known and/or is in use as an anti-NB agent, such asancitabine hydrochloride, doxorubicin hydrochloride, etoposide, orvincristine sulfate, or these agents in combination with one or more ofa different anti-NB TIC compound, such as a volume of one or more ofeach of the compounds listed below:

-   -   2.3-Dimethoxy-1.4-naphthoquinone,    -   Aklavine Hydrochloride,    -   Amodiaquin dihydrochloride dehydrate;    -   Amsacrine Hydrochloride;    -   Azaguanine-8;    -   beta-peltatin;    -   Camptothecine (S.+);    -   CGP-74514A hydrochloride;    -   Chelerythrine chloride;    -   Cholestan-3beta.5alpha.6beta-Triol;    -   Ciclopirox Olamine;    -   Clofazimine;    -   Colchicine;    -   Convallatoxin;    -   Crassin Acetate;    -   Crinamine;    -   Dequalinium analog. C-14 linker;    -   Dequalinium dichloride;    -   Digitoxin;    -   Digoxigenin;    -   Dihydrogambogic acid;    -   Dihydroouabain;    -   Erysolin;    -   Gambogic acid;    -   Mechlorethamine;    -   Meclizine hydrochloride;    -   MG 624;    -   Mitoxanthrone Hydrochloride;    -   Ouabain;    -   Oxybendazole;    -   Oxybendazole;    -   Paclitaxel;    -   Parthenolide;    -   Patulin;    -   Periplocymarin;    -   Peruvoside;    -   Primaquine diphosphate;    -   Quinacrine dihydrochloride;    -   Sanguinarine chloride; or Tomatine.

Example 20 Personalized Medicine Cancer Stem Cell Drug Kit

The present example demonstrates the utility of the present inventionfor providing a personalized medicine cancer stem cell drug kit.

In some embodiments, the kit will include a multi-well assay plate, suchas a standard 96-well assay plate. A volume of a potential anti-NBand/or anti-NB TIC compound/chemical entity will then be added to eachwell of a standard multi-well assay plate. In addition, the kit willinclude one or more wells to which no anti-NB or anti-NB TIC compoundhas been added, and will serve as the positive control in the assay.

A volume of NB TIC isolated from a patient being screened will then beadded to each well of the assay plate, along with a cell viabilityindicator agent, such as a cell viability indicator dye, Alamar Blue.The cells will be allowed to incubate for a period of time, after whichtime the intensity of the cell viability indicator agent will beassesed. The wells that demonstrate the greatest inhibition of cellproliferation or survival relative to the control well will be selectedfor use in treating the patient having NB.

In other embodiments, and as an added control for assessing potentialtoxicity to normal cells, the assay multi-well plate may include 2 ormore wells that will include a volume of each potential anti-NB compoundof interest. To one of each of these wells will be added a number of thepatients' isolated NB TIC, and to one or more of the wells will be addeda number of the patients' normal cells, such as human pediatricneural-crest derived stem cells from the dermis (i.e., SKPs). A cellviability indicator agent, such as the cell viability indicator dye,Alamar Blue, will then be added to each well, and the cells allowed toincubate. The intensity of the indicator agent will then be assessed asdescribed above. In this way, a potential anti-NB compound may beselected that causes the least amount of normal cell proliferationinhibiting activity.

In other embodiments, and as an added control for assessing potentialtoxicity to normal cells, the assay multi-well plate may include 2 ormore wells that will include a volume of each potential anti-NB compoundof interest. To one of each of these wells will be added a number of thepatients' isolated NB TIC, and to one or more of the wells will be addeda number of the patients' normal cells, such as human pediatricneural-crest derived stem cells from the dermis (i.e., SKPs). A cellviability indicator agent, such as the cell viability indicator dye,Alamar Blue, will then be added to each well, and the cells allowed toincubate. The intensity of the indicator agent will then be assessed asdescribed above. In this way, a potential anti-NB compound may beselected that causes the least amount of normal cell proliferationinhibiting activity.

In yet another aspect, the invention provides a kit for the testingand/or screening of a patient of interest's TICs from tumors such asleukemia, melanoma, brain, breast, and colon. Note that Tables 7, 11,14, and 17 identify parthenolide, a compound that specifically targetsNB TICs, as a compound previously identified to target human acutemyelogenous leukemia stem and progenitor cells (28), and thereforecompounds identified that target NB TICs can also target TICs from othertumors.

In this manner, a sample of biological tissue enriched for a populationof TICs from a patient of interest may be used to screen and/or identifya specific anti-TIC active agent or agents that are the most potentand/or active against a specific patient's TIC population, using theabove compounds.

Example 21 Dual Screening Method for Compounds having Activity forNeuroblastoma and Neuroblastoma Tumor Initiating Cells

The present example demonstrates the utility of the present invention asa dual screening method effective in the screening of a library ofcompounds and the identification of compounds for the treatment of NB,and compounds that are specifically cytostatic or cytotoxic toward NBTICs.

The screening method is a dual screening method because it employs bothnormal cells and NB TICs to asses the activity of a compound.

While virtually any normal cell line may be used as the normal cellpopulation in the assay, a particular normal cell line that may be usedare the FS90 and FS105 “normal” control cell lines (human Skin-derivedprecursors (SKPs)).

While virtually any NB TIC line may be used in the dual screen assay, aparticular NB TIC line that may be used in the assay is NB12, a stage IVNB cell line that was obtained from a patient having been heavilytreated for NB and having experienced multiple relapses of the disease.

Alternatively, the assay may be conducted wherein a NB patients' ownnormal tissue cells may be used as a control tissue in the screeningassay, such as a preparation of SKPs of the NB patient. In particular,these normal tissue cells are human pediatric neural crest-derived stemcells from the dermis (SKPs) of a patient, or derived from bone cells,nerve cells or muscle cells from the same patient.

Turning now to the diagram provided at FIG. 10, a sample of normaltissue cells is dissociated into spheres to provide a control celldissociated sample of cells; a sample of NB tumor cells is dissociatedinto spheres to provide a test cell dissociated sample of cells;depositing a number of cells from the control cell dissociated sample ofcells into a desired number of wells of a multi-well assay plate toprovide control cell wells; depositing the same number of cells from thetest cell dissociated sample of cells into a desired number of wells ofthe multi-well assay plate to provide test cell wells; adding a volumeof a potential anti-NB compound to each of said control cell wells andto each of said test cell wells to provide a loaded multi-well assayplate; incubating said loaded multi-well assay plate; adding a cellproliferation or survival indicator agent to each well of said loadedmulti well assay plate; and assessing indicator agent intensity in eachwell of said loaded multi-well assay plate, and selecting potentialanti-NB compounds that elicit a cell proliferation or survival indicatoragent intensity that is two (2) fold or more less intense than theindicator agent intensity observed in the control wells.

In some embodiments, the cell proliferation or survival indicator agentis a cell viability dye, such as alamarBlue®. In these embodiments, cellproliferation is assesed as with alamarBlue® intensity used in analamarBlue® assay. In application the alamarBlue® signal observed waslinear with time, there was minimal background, and there was lowvariability between wells and plates (CV 3.5-4.5%, Z>0.5), and there wasa greater tan 10-fold difference between control and backgroundfluorescence readings. In the trials run, the anti-NB compounds thatwere selected (“hits”) elicited a signal indicator intensity that wasshifted three (3) standard deviations from the mean signal indicatorintensity.

The screening assay may also include positive control compound wells,wherein a known anti-NB TIC or known anti-NB therapeutic agent, is addedto one or more wells containing control cells and to one or more wellscontaining NB cells. By way of example, such known anti-NB therapeuticagents are ancitabine hydrochloride, doxorubicin hydrochloride,etoposide, or vincristine sulfate. In this manner, each assay will haveits own positive control reference for assessing viability in the assayrun.

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference. Althoughthe present invention has been fully described in conjunction withseveral embodiments thereof with reference to the accompanying drawings,it is to be understood that various changes and modifications may beapparent to those skilled in the art. Such changes and modifications areto be understood as included within the scope of the present inventionas defined by the appended claims, unless they depart therefrom.

Bibliography

The references listed below as well as the references cited throughoutthe specification are incorporated herein by reference to the extentthat they supplement, explain, provide a background for or teachmethodology, techniques and/or compositions employed herein.

-   1. Brodeur G M. (2003), Nat Rev Cancer, 3:203-16.-   2. Maris J M. (2005), Curr Opin Pediatr, 17:7-13.-   3. van Limpt V., et al. (2005), Cancer Lett, 228:59-63.-   4. Pardal, R., et al. (2003), Nat Rev Cancer, 3:895-902.-   5. Beachy, P. A., et al. (2004), Nature, 432:324-31.-   6. Warner, J. K., et al. (2004), Oncogene, 23:7164.-   7. Hamburger, A. & Salmon, S. E. (1997), J Clin Invest, 60:846-54.-   8. Heppner, G. H. (1984), Cancer Res, 44:2259-65.-   9. Singh, S. K., et al. (2004), Oncogene, 23:7267-73.-   10. Al-Hajj, M. & Clarke, M. F. (2004), Oncogene, 23:7274-82.-   11. Lapidot T, et al. (1994), Nature, 367:645-8.-   12. Bonnet D, Dick JE. (1997), Nat Med, 3:730-7.-   13. Al-Hajj, M., (2003), Proc Natl Acad Sci USA, 100:3983-8.-   14. Singh, S. K. et al. (2004), Nature, 432:396-401.-   15. van Noesel M M, et al. (1997), Cancer, 80:834-43.-   16. Toma, J. G. et al. (2001), Nat Cell Biol, 3:778-84.-   17. Fernandes K J, et al. (2004), Nat Cell Biol, 6:1082-93.-   18. Toma J G, et al. (2005), Stem Cells, 23:727-37.-   19. Christiansen J H, (2000), Curr Opin Cell Biol, 12:719-24.-   20. Ambros P F, et al. (2003), Cancer Lett, 197:29-34.-   21. Miettinen M, et al. (1998), Am J Surg Pathol, 22:327-32.-   22. LaBrosse E H, et al. (1976), J Natl Cancer Inst, 57:633-8.-   23. Barnabe-Heider F, Miller F D. (2003), J Neurosci, 23:5149-60.-   24. Marsh H N, et al. (2003), J Cell Biol, 163:999-1010.-   25. Lo Piccolo M S, Cheung N K, Cheung I Y. (2001), Cancer,    92:924-31.-   26. Fernandes K J, et al. (2006), Exp Neurol, 201:32-48.-   27. Hafer R, et al. (1999), J Neuroimmunol, 96:201-6.-   28. Khanna C, et al. (2002), In Vivo, 16:77-85.-   29. Nakagawara A, Ohira M. (2004), Cancer Lett, 204:213-24.-   30. Ohira M, et al. (2005), Cancer Cell, 7:337-50.-   31. Weiss W A, et al. (1997), EMBO J, 16:2985-95.-   32. ElShamy W M, Fridvall L K, Ernfors P. (1998), Neuron,    21:1003-15.-   33. Lasorella A, et al (2002) Cancer Res, 62:301-6.-   34. Valsesia-Wittmann S, et al. (2004), Cancer Cell, 6:625-30.-   35. Dubreuil V, et al. (2000), Development, 127:5191-201.-   36. Pattyn A, (2000), Mol Cell Neurosci, 15:235-43.-   37. Pozniak C D, (2000), Science, 289:304-6.-   38. Casciano I, et al. (2002), Cell Death Differ, 9:246-51.-   39. Matsumoto K, et al. (1995), Cancer Res, 55:1798-806-   40. Jaboin J, et al. (2002), Cancer Res, 62:6756-63.-   41. Kaplan D R, et al. (1993), Neuron, 11:321-31.-   42. Lucarelli E, et al. (1997), Eur J Cancer, 33:2068-70.-   43. Lavoie J F, et al. (2005), J Biol. Chem., 280:29199-207.-   44. Wartiovaara K, et al. (2002), J Neurosci, 22:815-24.-   45. Atwal J K, et al (2000), Neuron, 27:265-77.-   46. Toma J G, et al. (2000), J Neurosci, 20:7648-56.-   47. Ellis J, Yao S. (2005), Curr Gene Ther, 5:367-73.-   48. Ellis J., (2005), Human Gene Ther, 16:1241-6.-   49. Mckenzie I A, et al. (2006), J Neurosci, 26:6651-60.-   50. Torkin R, et al. (2005), Mol Cancer Ther, 4:1-11.-   51. Barnabe-Heider F, et al. (2005), Neuron, 48: 253-65.-   52. U.S. Pat. No. 6,787,355—Miller, et al. (2004).-   53. Guzman, M L., et al., (2005), Blood, 105(11): 4163-9.-   54. Singh S K, et al. (2003), Cancer Res, 63:5821-28.-   55. Reynolds B A & Weiss S. (1996), Dev Biol; 175:1-13.-   56. Clarke M F, et al., (2006), Cancer Res, 66:9339-44.-   57. Fang D, et al. (2005), Cancer Res; 65:9328-37.-   58. Nagai J, et al. (2000), J Pediatr Hematol Oncol, 22:20-6.-   59. Bata-Csorgo Z, et al. (1993), J Exp Med 8:1271-81.-   60. Akashi T, et al. (1994), Virchows Arch, 425:399-406.-   61. Ponti D, et al. (2005), Cancer Res, 65:5506-11.-   62. Choi H S, et al. (2005), Pediatr Blood Cancer, 45:68-71.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. A method foridentifying compounds having anti-neuroblastoma tumor initiating cellactivity comprising: preparing an enriched preparation of neuroblastomatumor cells from a patient having neuroblastoma to provide an enrichedtest cell dissociated sample of cells; depositing a number of cells fromthe enriched test cell dissociated sample of cells into a desired numberof wells of the multi-well assay plate to provide test cell wells andtwo or more control wells; adding a volume of a potentialanti-neuroblastoma compound to each of said test cell wells to provide aloaded multi-well assay plate; adding a cell proliferation indicatoragent to each well of said loaded multi-well assay plate and incubatingsaid loaded multi-well assay plate; comparing indicator agent intensityin each well of said test cell wells to said control wells; andselecting potential anti-neuroblastoma compounds that elicit a cellproliferation indicator agent intensity that is two (2) fold or lessintense than the indicator agent intensity observed in a control cellwell.
 6. The method of claim 5 wherein the cell proliferation indicatoragent is a cell viability dye.
 7. The method of claim 6 wherein the cellviability dye is Alamar Blue or another cell viability dye.
 8. A methodfor selecting compounds having anti-neuroblastoma tumor-initiating cellactivity comprising: preparing a culture of cells enriched forneuroblastoma tumor initiating cells to provide an enriched test cellculture; providing a culture of cells other than neuroblastomatumor-initiating cells to provide a control cell culture; providing afirst test volume of a compound from a compound library of interest to asample of the test cell culture and a second test volume of the compoundof interest to a sample of the control cell culture from the compoundlibrary of interest; assessing cell activity in the test cell culturesample and in the control cell culture sample; and selecting a compoundthat reduces the activity of the cells in the test cell culture sampleand that does not reduce the activity of the cells in the control cellculture sample.
 9. The method of claim 8 wherein an indicator dye ofcell viability is added to each well.
 10. The method of claim 9 whereinthe indicator dye of cell viability is Alamar blue.
 11. The method ofclaim 8 further comprising one or more wells having a knownanti-neuroblastoma tumor initiating cell therapeutic agent or knownanti-neuroblastoma therapeutic agent.
 12. The method of clam 11 whereinthe known anti-neuroblastoma tumor initiating cell therapeutic agent isancitabine hydrochloride, doxorubicin hydrochloride, etoposide, orvincristine sulfate.
 13. A kit for selecting an anti-neuroblastoma tumorinitiating cell preparation for a patient, said kit comprising: an assayplate that includes a plurality of wells, each well of said assay platebeing suitable for containing an anti-neuroblastoma tumor initiatingcell pharmacologically active agent and a volume of cells; a volume of apanel of individual anti-neuroblastoma tumor initiating cellpharmacologically active agents, wherein 2 or more of said wells eachcontain a volume of each anti-neuroblastoma tumor initiating cellpharmacologically active agent.
 14. The kit of claim 13 further definedas comprising a 96-well assay plate.
 15. The kit of claim 13 wherein thepanel of individual anti-neuroblastoma tumor initiating cellpharmacologically active agents comprises a volume of one or more ofeach of the compounds listed below: 2.3-Dimethoxy-1.4-naphthoquinone,Aklavine Hydrochloride, Amodiaquin dihydrochloride dehydrate; AmsacrineHydrochloride; Azaguanine-8; beta-peltatin; Camptothecine (S.+);CGP-74514A hydrochloride; Chelerythrine chloride;Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine; Clofazimine;Colchicine; Convallatoxin; Crassin Acetate; Crinamine; Dequaliniumanalog. C-14 linker; Dequalinium dichloride; Digitoxin; Digoxigenin;Dihydrogambogic acid; Dihydroouabain; Erysolin; Gambogic acid;Mechlorethamine; Meclizine hydrochloride; MG 624; MitoxanthroneHydrochloride; Ouabain; Oxybendazole; Oxybendazole; Paclitaxel;Parthenolide; Patulin; Periplocymarin; Peruvoside; Primaquinediphosphate; Quinacrine dihydrochloride; Sanguinarine chloride; orTomatine.
 16. The kit of claim 15 wherein the panel of individualanti-neuroblastoma tumor initiating cell pharmacologically active agentscomprises a volume of ancitabine hydrochloride, doxorubicinhydrochloride, etoposide, or vincristine sulfate.
 17. The kit of claim13 wherein the kit further comprises an instructional manual.
 18. A kitfor screening a patient of interest to identify an appropriateanti-tumor cell agent, said kit comprising: an assay plate comprising aplurality of wells suitable for containing a volume of one of eachcompound listed below: 2.3-Dimethoxy-1.4-naphthoquinone, AklavineHydrochloride, Amodiaquin dihydrochloride dehydrate; AmsacrineHydrochloride; Azaguanine-8; beta-peltatin; Camptothecine (S.+);CGP-74514A hydrochloride; Chelerythrine chloride;Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine; Clofazimine;Colchicine; Convallatoxin; Crassin Acetate; Crinamine; Dequaliniumanalog. C-14 linker; Dequalinium dichloride; Digitoxin; Digoxigenin;Dihydrogambogic acid; Dihydroouabain; Erysolin; Gambogic acid;Mechlorethamine; Meclizine hydrochloride; MG 624; MitoxanthroneHydrochloride; Ouabain; Oxybendazole; Oxybendazole; Paclitaxel;Parthenolide; Patulin; Periplocymarin; Peruvoside; Primaquinediphosphate; Quinacrine dihydrochloride; Sanguinarine chloride; orTomatine, and wherein one or more assay plate wells is a positivecontrol well that contains ancitabine hydrochloride, doxorubicinhydrochloride, etoposide, or vincristine sulfate; and an instructionalsheet describing a method by which an enriched preparation ofneuroblastoma tumor initiating cells may be isolated from the patient ofinterest.
 19. The kit of claim 18 further comprising positive controlcompound wells that are absent an anti-tumor cell agent.
 20. The kit ofclaim 18 further comprising a cell viability indicator dye.
 21. A kitfor screening a patient of interest to identify an appropriateanti-tumor initiating cell active agent for tumor-initiating cells ofleukemia, melanoma, breast cancer, brain cancer, or colon cancer, saidkit comprising: an assay plate comprising a plurality of wells suitablefor containing an anti-tumor initiating cell compound, wherein each wellcontains a volume of one of each compound listed below:2.3-Dimethoxy-1,4-naphtho quinone, Aklavine Hydrochloride, Amodiaquindihydrochloride dehydrate; Amsacrine Hydrochloride; Azaguanine-8;beta-peltatin; Camptothecine (S.+); CGP-74514A hydrochloride;Chelerythrine chloride; Cholestan-3beta.5alpha.6beta-Triol; CiclopiroxOlamine; Clofazimine; Colchicine; Convallatoxin; Crassin Acetate;Crinamine; Dequalinium analog. C-14 linker; Dequalinium dichloride;Digitoxin; Digoxigenin; Dihydrogambogic acid; Dihydroouabain; Erysolin;Gambogic acid; Mechlorethamine; Meclizine hydrochloride; MG 624;Mitoxanthrone Hydrochloride; Ouabain; Oxybendazole; Oxybendazole;Paclitaxel; Parthenolide; Patulin; Periplocymarin; Peruvoside;Primaquine diphosphate; Quinacrine dihydrochloride; Sanguinarinechloride; or Tomatine, and wherein one or more assay plate wells is apositive control well that contains ancitabine hydrochloride,doxorubicin hydrochloride, etoposide, or vincristine sulfate; and aninstructional sheet describing a method by which an enriched preparationof tumor initiating cells may be isolated from the patient of interest.22. The kit of claim 21 further comprising positive control compoundwells that are absent an anti-tumor initiating cell active agent. 23.The kit of claim 21 further comprising a cell viability indicator dye.